Selective Cerebrospinal Fluid Hypothermia: Bioengineering Development and In Vivo Study of an Intraventricular Cooling Device (V-COOL).

Hypothermia is a promising therapeutic strategy for severe vasospasm and other types of non-thrombotic cerebral ischemia, but its clinical application is limited by significant systemic side effects. We aimed to develop an intraventricular device for the controlled cooling of the cerebrospinal fluid, to produce a targeted hypothermia in the affected cerebral hemisphere with a minimal effect on systemic temperature. An intraventricular cooling device (acronym: V-COOL) was developed by in silico modelling, in vitro testing, and in vivo proof-of-concept application in healthy Wistar rats (n = 42). Cerebral cortical temperature, rectal temperature, and intracranial pressure were monitored at increasing flow rate (0.2 to 0.8 mL/min) and duration of application (10 to 60 min). Survival, neurological outcome, and MRI volumetric analysis of the ventricular system were assessed during the first 24 h. The V-COOL prototyping was designed to minimize extra-cranial heat transfer and intra-cranial pressure load. In vivo application of the V-COOL device produced a flow rate-dependent decrease in cerebral cortical temperature, without affecting systemic temperature. The target degree of cerebral cooling (- 3.0 °C) was obtained in 4.48 min at the flow rate of 0.4 mL/min, without significant changes in intracranial pressure. Survival and neurological outcome at 24 h showed no significant difference compared to sham-treated rats. MRI study showed a transient dilation of the ventricular system (+ 38%) in a subset of animals. The V-COOL technology provides an effective, rapid, selective, and safe cerebral cooling to a clinically relevant degree of - 3.0 °C.

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.

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.

Positive impact of dynamic seeding of mesenchymal stem cells on bone-like biodegradable scaffolds with increased content of calcium phosphate nanoparticles.

One of the main aims of bone tissue engineering, regenerative medicine and cell therapy is development of an optimal artificial environment (scaffold) that can trigger a favorable response within the host tissue, it is well colonized by resident cells of organism and ideally, it can be in vitro pre-colonized by cells of interest to intensify the process of tissue regeneration. The aim of this study was to develop an effective tool for regenerative medicine, which combines the optimal bone-like scaffold and colonization technique suitable for cell application. Accordingly, this study includes material (physical, chemical and structural) and in vitro biological evaluation of scaffolds prior to in vivo study. Thus, porosity, permeability or elasticity of two types of bone-like scaffolds differing in the ratio of collagen type I and natural calcium phosphate nanoparticles (bCaP) were determined, then analyzes of scaffold interaction with mesenchymal stem cells (MSCs) were performed. Simultaneously, dynamic seeding using a perfusion bioreactor followed by static cultivation was compared with standard static cultivation for the whole period of cultivation. In summary, cell colonization ability was estimated by determination of cell distribution within the scaffold (number, depth and homogeneity), matrix metalloproteinase activity and gene expression analysis of signaling molecules and differentiation markers. Results showed, the used dynamic colonization technique together with the newly-developed collagen-based scaffold with high content of bCaP to be an effective combined tool for producing bone grafts for bone implantology and regenerative medicine.

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.

Assembly of Tissue-Engineered Blood Vessels with Spatially Controlled Heterogeneities.

Tissue-engineered human blood vessels may enable in vitro disease modeling and drug screening to accelerate advances in vascular medicine. Existing methods for tissue-engineered blood vessel (TEBV) fabrication create homogenous tubes not conducive to modeling the focal pathologies characteristic of certain vascular diseases. We developed a system for generating self-assembled human smooth muscle cell (SMC) ring units, which were fused together into TEBVs. The goal of this study was to assess the feasibility of modular assembly and fusion of ring building units to fabricate spatially controlled, heterogeneous tissue tubes. We first aimed to enhance fusion and reduce total culture time, and determined that reducing ring preculture duration improved tube fusion. Next, we incorporated electrospun polymer ring units onto tube ends as reinforced extensions, which allowed us to cannulate tubes after only 7 days of fusion, and culture tubes with luminal flow in a custom bioreactor. To create focal heterogeneities, we incorporated gelatin microspheres into select ring units during self-assembly, and fused these rings between ring units without microspheres. Cells within rings maintained their spatial position along tissue tubes after fusion. Because tubes fabricated from primary SMCs did not express contractile proteins, we also fabricated tubes from human mesenchymal stem cells, which expressed smooth muscle alpha actin and SM22-α. This work describes a platform approach for creating modular TEBVs with spatially defined structural heterogeneities, which may ultimately be applied to mimic focal diseases such as intimal hyperplasia or aneurysm.

Dry versus hydrated collagen scaffolds: are dry states representative of hydrated states?

Collagen composite scaffolds have been used for a number of studies in tissue engineering. The hydration of such highly porous and hydrophilic structures may influence mechanical behaviour and porosity due to swelling. The differences in physical properties following hydration would represent a significant limiting factor for the seeding, growth and differentiation of cells in vitro and the overall applicability of such hydrophilic materials in vivo. Scaffolds based on collagen matrix, poly(DL-lactide) nanofibers, calcium phosphate particles and sodium hyaluronate with 8 different material compositions were characterised in the dry and hydrated states using X-ray microcomputed tomography, compression tests, hydraulic permeability measurement, degradation tests and infrared spectrometry. Hydration, simulating the conditions of cell seeding and cultivation up to 48 h and 576 h, was found to exert a minor effect on the morphological parameters and permeability. Conversely, hydration had a major statistically significant effect on the mechanical behaviour of all the tested scaffolds. The elastic modulus and compressive strength of all the scaffolds decreased by ~95%. The quantitative results provided confirm the importance of analysing scaffolds in the hydrated rather than the dry state since the former more precisely simulates the real environment for which such materials are designed.

Functional Tricuspid Regurgitation Model in a Beating Heart Platform.

Currently, clinicians are seeking new, minimally invasive treatment options for functional tricuspid regurgitation (FTR). Challenging tricuspid complexity requires the evaluation of the treatment techniques in adequate and realistic preclinical scenario. The purpose of this article is to describe the design and functional assessment of a novel passive beating heart model of the pulmonary circulation with the possibility to tightly control FTR. The model housed porcine hearts actuated by a volumetric pump that cyclically pressurized the right ventricle. The in-vitro FTR model exploited the tendency of the ventricle to dilate under pressure. The dilation entailed papillary muscles displacement and valve annulus enlargement, thus inducing tricuspid valve insufficiency. Employment of constraint bands allowed to restore valve competency. The system provided consistent replication of the main determinants of the pulmonary hemodynamics in a wide range of working conditions. The experimental model of FTR was reliable, easily controllable, and showed good stability-over-time. Echocardiography and fiberscope imaging provided a unique opportunity to investigate valve dynamics. These features make the platform suitable for realistic training purposes and testing of the upcoming FTR therapies.

Full Mimicking of Coronary Hemodynamics for Ex-Vivo Stimulation of Human Saphenous Veins.

After coronary artery bypass grafting, structural modifications of the saphenous vein wall lead to lumen narrowing in response to the altered hemodynamic conditions. Here we present the design of a novel ex vivo culture system conceived for mimicking central coronary artery hemodynamics, and we report the results of biomechanical stimulation experiments using human saphenous vein samples. The novel pulsatile system used an aortic-like pressure for forcing a time-dependent coronary-like resistance to obtain the corresponding coronary-like flow rate. The obtained pulsatile pressures and flow rates (diastolic/systolic: 80/120 mmHg and 200/100 mL/min, respectively) showed a reliable mimicking of the complex coronary hemodynamic environment. Saphenous vein segments from patients undergoing coronary artery bypass grafting (n = 12) were subjected to stimulation in our bioreactor with coronary pulsatile pressure/flow patterns or with venous-like perfusion. After 7-day stimulation, SVs were fixed and stained for morphometric evaluation and immunofluorescence. Results were compared with untreated segments of the same veins. Morphometric and immunofluorescence analysis revealed that 7 days of pulsatile stimulation: (i) did not affect integrity of the vessel wall and lumen perimeter, (ii) significantly decreased both intima and media thickness, (iii) led to partial endothelial denudation, and (iv) induced apoptosis in the vessel wall. These data are consistent with the early vessel remodeling events involved in venous bypass adaptation to arterial flow/pressure patterns. The pulsatile system proved to be a suitable device to identify ex vivo mechanical cues leading to graft adaptation.

Design of a simple coronary impedance simulator for the in vitro study of the complex coronary hemodynamics.

Several novel approaches were recently developed to treat aortic root pathologies. The alteration induced by some of these approaches to the biomechanics of the aortic root could possibly affect the coronary perfusion, compromising the procedural outcome. In this scenario, the need to replicate in vitro the coronary flow pattern in physiological and pathological conditions is becoming crucial for the functional assessment of novel devices and techniques. This article describes the design of an easy-to-use, left-and-right coronary impedance simulator, coupled with native aortic roots for in vitro pulsatile tests. Experiments were performed in order to assess the performances of the coronary impedance simulator when coupled with healthy aortic valves (cardiac output: 3.8 ± 0.26 l min-1; mean systemic pressure: 95 ± 1.3 mmHg; mean coronary flow rate: 272 ± 13.4 ml min-1) or with regurgitant valves (cardiac output: 1.9 ± 0.24 l min-1; mean systemic pressure of 45 ± 3.3 mmHg; mean coronary flow rate:149 ± 21.9 ml min-1). The acute systemic response to valve regurgitation was also replicated, with increased beat rate and afterload, aimed at restoring the systemic pressure (cardiac output: 2.5 ± 0.23 l min-1; mean systemic pressure of 109 ± 6.1 mmHg; mean coronary flow rate: 262 ± 35.5 ml min-1). In the test conditions, the system was able to replicate in vitro the main determinants of the coronary circulation with physiological left/right coronary flow rate repartition, and a realistic interaction between coronary and systemic hemodynamics. The coronary simulator appears to be a suitable platform to study and optimize the interactions between novel approaches to aortic valve pathology and the coronary perfusion.

In vitro assessment of mitral valve function in cyclically pressurized porcine hearts.

Recent approaches to the in vitro experimental study of cardiac fluid mechanics involve the use of whole biological structures to investigate in the lab novel therapeutic approaches for the treatment of heart pathologies. To enhance reliability and repeatability, the influence of the actuation strategy of the experimental apparatuses on the biomechanics of biological structures needs to be assessed. Using echography and intracardiac high-speed imaging, we compared the mitral valve (MV) anatomo-functional features (coaptation areas/lengths, papillary muscles-valvular plane distances) in two passive-beating-heart mock loops with internal (IPML) or external (EPML) pressurization of the ventricular chamber. Both apparatuses showed fluid dynamic conditions that closely resembled the physiology. The MVs analyzed in the EPML presented coaptation areas and lengths that were systematically higher, and exhibited greater variability from early-to peak-systole, as compared to those in the IPML. Moreover, in the EPML, the MV leaflets exhibited a convexity with high curvature toward the atrium. With the IPML, MV coaptation lengths ranged similar to available clinical data and the papillary muscles-valve plane distances were more stable throughout systole. In conclusion, both the apparatuses allow for reproducing in vitro the left heart hemodynamics, in terms of flow rates and pressures, with proper mitral valve continence. Results suggest that the IPML is more suitable for replicating the physiological MV functioning, while the EPML may have more potential as a model for the study of MV pathologies.

Human Saphenous Vein Response to Trans-wall Oxygen Gradients in a Novel Ex Vivo Conditioning Platform.

Autologous saphenous veins are commonly used for the coronary artery bypass grafting even if they are liable to progressive patency reduction, known as 'vein graft disease'. Although several cellular and molecular causes for vein graft disease have been identified using in vivo models, the metabolic cues induced by sudden interruption of vasa vasorum blood supply have remained unexplored. In the present manuscript, we describe the design of an ex vivo culture system allowing the generation of an oxygen gradient between the luminal and the adventitial sides of the vein. This system featured a separation between the inner and the outer vessel culture circuits, and integrated a purpose-developed de-oxygenator module enabling the trans-wall oxygen distribution (high oxygen level at luminal side and low oxygen level at the adventitial side) existing in arterialized veins. Compared with standard cultures the bypass-specific conditions determined a significant increase in the proliferation of cells around adventitial vasa vasorum and an elevation in the length density of small and large caliber vasa vasorum. These results suggest, for the first time, a cause-effect relationship between the vein adventitial hypoxia and a neo-vascularization process, a factor known to predispose the arterialized vein conduits to restenosis.

A compact and automated ex vivo vessel culture system for the pulsatile pressure conditioning of human saphenous veins.

Saphenous vein (SV) graft disease represents an unresolved problem in coronary artery bypass grafting (CABG). After CABG, a progressive remodelling of the SV wall occurs, possibly leading to occlusion of the lumen, a process termed 'intima hyperplasia' (IH). The investigation of cellular and molecular aspects of IH progression is a primary end-point toward the generation of occlusion-free vessels that may be used as 'life-long' grafts. While animal transplantation models have clarified some of the remodelling factors, the pathology of human SV is far from being understood. This is also due to the lack of devices able to reproduce the altered mechanical load encountered by the SV after CABG. This article describes the design of a novel ex vivo vein culture system (EVCS) capable of replicating the altered pressure pattern experienced by SV after CABG, and reports the results of a preliminary biomechanical conditioning experimental campaign on SV segments. The EVCS applied a CAGB-like pressure (80-120 mmHg) or a venous-like perfusion (3 ml/min, 5 mmHg) conditioning to the SVs, keeping the segments viable in a sterile environment during 7 day culture experiments. After CABG-like pressure conditioning, SVs exhibited a decay of the wall thickness, an enlargement of the luminal perimeter, a rearrangement of the muscle fibres and partial denudation of the endothelium. Considering these preliminary results, the EVCS is a suitable system to study the mechanical attributes of SV graft disease, and its use, combined with a well-designed biological protocol, may be of help in elucidating the cellular and molecular mechanisms involved in SV graft disease.

Feasibility of pig and human-derived aortic valve interstitial cells seeding on fixative-free decellularized animal pericardium.

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.

Adventitial vessel growth and progenitor cells activation in an ex vivo culture system mimicking human saphenous vein wall strain after coronary artery bypass grafting.

Saphenous vein graft disease is a timely problem in coronary artery bypass grafting. Indeed, after exposure of the vein to arterial blood flow, a progressive modification in the wall begins, due to proliferation of smooth muscle cells in the intima. As a consequence, the graft progressively occludes and this leads to recurrent ischemia. In the present study we employed a novel ex vivo culture system to assess the biological effects of arterial-like pressure on the human saphenous vein structure and physiology, and to compare the results to those achieved in the presence of a constant low pressure and flow mimicking the physiologic vein perfusion. While under both conditions we found an activation of Matrix Metallo-Proteases 2/9 and of microRNAs-21/146a/221, a specific effect of the arterial-like pressure was observed. This consisted in a marked geometrical remodeling, in the suppression of Tissue Inhibitor of Metallo-Protease-1, in the enhanced expression of TGF-ß1 and BMP-2 mRNAs and, finally, in the upregulation of microRNAs-138/200b/200c. In addition, the veins exposed to arterial-like pressure showed an increase in the density of the adventitial vasa vasorum and of cells co-expressing NG2, CD44 and SM22α markers in the adventitia. Cells with nuclear expression of Sox-10, a transcription factor characterizing multipotent vascular stem cells, were finally found in adventitial vessels. Our findings suggest, for the first time, a role of arterial-like wall strain in the activation of pro-pathologic pathways resulting in adventitial vessels growth, activation of vasa vasorum cells, and upregulation of specific gene products associated to vascular remodeling and inflammation.

A lumped-parameter approach for designing a novel pulsatile bioreactor for ex-vivo studies of human saphenous vein remodeling.

After coronary artery bypass grafting (CABG), the hemodynamic conditions experienced by the saphenous veins are similar to those experienced by coronary arteries: pulsatile pressure (80-120 mmHg), pulsatile flow (mean flow rate of 250 ml/min), and elevated shear stress (1-7 Pa). Here we present a novel pulsatile platform for studying the human saphenous vein early remodeling events after CABG. The system was designed in order to apply CABG-like pressure/flow stimulation patterns to the hosted human saphenous vein segments, i.e. a pulsatile pressure oscillating between a diastolic minimum and a systolic maximum in counter-phase with a pulsatile flow rate. Functional tests revealed good fitting of simulated and measured tracings and the ability of the pulsatile platform to mimic the complexity of the coronary hemodynamic environment with good fidelity in comparison with state-of-the-art devices. This system will enable us to study the biological response of human saphenous veins to CABG conditioning ex vivo, in currently ongoing experiments.

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.

Tools and procedures for ex vivo vein arterialization, preconditioning and tissue engineering: a step forward to translation to combat the consequences of vascular graft remodeling.

The present contribution reviews recent progress in bioengineering approaches used to mimic arterial hemodynamic conditions in vascular grafts and vessel substitutes used in vascular surgery. While implantation of vascular bypasses is still the primary option for cardiac and vascular surgeons to recover blood perfusion in cardiac and peripheral ischemic tissues, effective techniques to reduce the impact of post-grafting vascular remodeling are insufficient. In our view, the design of specific bioreactors to perform vascular conditioning with complex stimulation patterns will provide valuable tools for comprehensive molecular analysis of vessel arterialization process. In addition, this approach will allow the future design of refined protocols to perform pre-conditioning of natural vessels, reseeding of human or animalderived decellularized vascular grafts or, finally, derivation of fully engineered arterial-compliant substitutes, with a reduced remodeling impact.