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.

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.

Our experience shows that using bioengineering approaches facilitates the understanding of vascular physio-pathological mechanisms and, in perspective, will speed up the development of new life-saving treatments. The use of human samples, particularly operating room-derived samples, which would have been otherwise discarded, is a very valuable approach. In line with the 3Rs principles, this methodology is worth the cost of being set up and managed, wherever and whenever possible

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-β-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-β-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.

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.

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.

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.

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.

According to World Health Organization(WHO), 3.2 billion people are at risk for malaria. In 2015, 212 million new cases and 429000 deaths were estimated [1, 2]. Despite treatment in the early stage of the disease is usually very effective, conventional diagnostic tests via optical microscopy examination of thick and thin blood smears are unsuitable for an effective screening of the population. On the other hand, the over-treatment of the disease due to the large percentage of false positives in currently available rapid diagnostic tests (RDTs) may increase the risk of drug resistance. In this scenario, there is a strong need of novel RTDs with (i) the same sensitivity of the gold standard (optical microscopy examination) and (ii) a reduced number of false positives. To fulfill the last requirement, a real improvement would be to move from the detection of antigens or antibodies, which can be hardly washed out in a patient living in an endemic zone even after many weeks from the last malaria episode, to the quantification of infected red blood cells in a blood smear (i-RBC). This essentially means to go back to the concept of gold standard tests, with the additional requirement of integrating i-RBC counting in lab-on-chip platforms suitable for low-cost, rapid and on-site wide screening of the population in endemic zones. It is well known that i-RBCs display a paramagnetic behavior with respect to blood plasma, so that they can be separated from healthy ones and other corpuscles in a high magnetic field gradient. [3] This is due to the fact that, during the intra-erythrocytic development, the parasite degrades hemoglobin into free heme. This molecule, highly toxic to the parasite, is converted in an insoluble form, known as hemozoin or malaria pigment, which crystallizes into paramagnetic nanocrystals found both within the i-RBCs and free in the blood, after RBCs lysis. Of course, both the concentration of free hemozoin crystals and i-RBCs can be used for the determination of the parasitemia. In this paper we present an on-chip magnetophoretic platform for the separation and concentration of i-RBC and hemozoin nanocrystals on pre-defined areas of a chip. This is a pre-requisite for the quantification of the relative percentage of i-RBC with respect to healthy ones (parasitemia) with high sensitivity. The blood drop is placed on a glass substrate, which is then put in close contact to the surface of a chip with Nickel micropillars, at a distance defined by an outer ring which defines also the volume of the cell where magnetophoretic separation takes place. The chip is placed face-down, so that magnetic attraction towards the nickel pillars, in the macroscopic field gradient produced by an external system of permanent magnets, opposes the gravity. In this configuration, i-RBCs and hemozoin crystals are attracted upwards, towards the micropillars, while non-infected erythrocytes and the other blood cells (i.e. white blood cells and platelets) sediment towards the glass substrate. Our design allows to obtain a macroscopic field gradient as high as 1 × 10 15 A 2 /m 3 up to a distance of 500 micron from the chip surface, strong enough to overcome gravity and attract i-RBCs and hemozoin crystals towards the chip surface. The chip consists of an array of Ni pillars, with 20-30 micron diameter and 20 micron height, fabricated by electroplating and arranged on a hexagonal closed packed lattice. In close proximity to the chip surface, Ni pillars produce a much stronger field gradient, up to 3 × 10 16 A 2 /m 3 at a few microns from their surface, which concentrate the i-RBCs and hemozoin crystals. We have tested this system using RBCs from bovine blood, treated with NaNO 2 in order to induce the transformation of hemoglobin into paramagnetic meta-hemoglobin. [4] In this way, we obtained suspensions in PBS of RBCs mimicking i-RBCs, suitable for experiments of capture. In figure 1 we report optical images from experiments performed in a direct configuration, where gravity and magnetic forces act in the same direction. Untreated RBCs (ut-RBCs) and treated ones with NaNO 2 (t-RBCs) were stained with a red fluorophore to improve the quality of optical images. As evident from Figure 1, in case of t-RBCs we observed a capture efficiency of 100% by magnetic pillars in a lattice with center to center spacing of 80 microns. For hemozoin crystals the capture is even easier, because of their much larger volume susceptibility, 3.4 × 10 -4 to be compared with 3.9 × 10 -6 in case of i-RBCs. These results pave the way to the use of our magnetic chips as active slides for the magnetophoretic separation and concentration of malaria markers, both i-RBCs and hemozoin crystals, in well-defined areas of the chips where quantification can be performed with high sensitivity.

In the past decades, vascular tissue engineering has made great strides towards bringing engineered vascular tissues to the clinics and, in parallel, obtaining in-lab tools for basic research. Herein, we propose the design of a novel dual-mode bioreactor, useful for the fabrication (construct mode) and in vitro stimulation (culture mode) of collagen-based tubular constructs. Collagen-based gels laden with smooth muscle cells (SMCs) were molded directly within the bioreactor culture chamber. Based on a systematic characterization of the bioreactor culture mode, constructs were subjected to 10% cyclic strain at 0.5 Hz for 5 days. The effects of cyclic stimulation on matrix re-arrangement and biomechanical/viscoelastic properties were examined and compared vs. statically cultured constructs. A thorough comparison of cell response in terms of cell localization and expression of contractile phenotypic markers was carried out as well. We found that cyclic stimulation promoted cell-driven collagen matrix bi-axial compaction, enhancing the mechanical strength of strained samples with respect to static controls. Moreover, cyclic strain positively affected SMC behavior: cells maintained their contractile phenotype and spread uniformly throughout the whole wall thickness. Conversely, static culture induced a noticeable polarization of cell distribution to the outer rim of the constructs and a sharp reduction in total cell density. Overall, coupling the use of a novel dual-mode bioreactor with engineered collagen-gel-based tubular constructs demonstrated to be an interesting technology to investigate the modulation of cell and tissue behavior under controlled mechanically conditioned in vitro maturation.

Background: Despite the preferred application of arterial conduits, the greater saphenous vein (SV) remains indispensable for bypass grafting, especially in multi-vessel coronary artery disease. Early remodeling induced by altered wall mechanics has been recognized to play a key role in SV graft disease. The mechanism remains, however, unknown. Aim: To investigate mechanical factors involved in early graft remodeling, we characterized SV-derived smooth muscle cells (SMCs) after both ex vivo coronary-like mechanical stimulation of SV segments and in vitro unidirectional strain. Methods: SV segments from patients receiving coronary artery bypass grafts were stimulated in a custom-made coronary pulse-duplicator bioreactor. After 7 (n=6) or 14 (n=5) days, stretched and control SVs were fixed and stained for immunofluorescence. Additionally, SMCs isolated from SVs of 7 patients undergoing saphenectomy were subjected to uniaxial cyclic strain (10% elongation, 1 Hz) for 24 or 72 hours using a Flexcell platform. SMCs analysis was performed by western blotting and mass spectrometry-based secretome analysis. Results: Coronary stimulation elevated apoptosis of SV medial cells after 7 days, and consistently reduced the percentage of cells positive for contractile markers α-SMA and calponin. Conversely, synthetic phenotype marker tropomyosin-4 (TM4) and early contractile marker SM22α were elevated at T14. Mesenchymal marker CD44 was markedly upregulated in cells populating the media after 14 days of stimulation. In accordance, strained SMCs displayed decreased α-SMA and SM22α, and increased TM4 protein expression after 72h. Analysis of the supernatant showed a significant increase of plasminogen activator inhibitor-1 and thrombospondin 1. Conclusions: Mechanical stimulation of SVs leads to apoptosis of medial cells and a decrease of contractile SMC markers, followed by repopulation with cells expressing the mesenchymal marker CD44. In addition, unidirectional strain induces a switch of SMC phenotype and secretion of proteins related to vascular remodeling. We are currently investigating whether CD44 + cells derive from SMCs undergoing phenotypic switch, or from progenitor cells localized in the adventitia.

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.