A Microfluidic Platform for Cavitation-Enhanced Drug Delivery.

An endothelial-lined blood vessel model is obtained in a PDMS (Polydimethylsiloxane) microfluidic system, where vascular endothelial cells are grown under physiological shear stress, allowing -like maturation. This experimental model is employed for enhanced drug delivery studies, aimed at characterising the increase in endothelial permeability upon microbubble-enhanced ultrasound-induced (USMB) cavitation. We developed a multi-step protocol to couple the optical and the acoustic set-ups, thanks to a 3D-printed insonation chamber, provided with direct optical access and a support for the US transducer. Cavitation-induced interendothelial gap opening is then analysed using a customised code that quantifies gap area and the relative statistics. We show that exposure to US in presence of microbubbles significantly increases endothelial permeability and that tissue integrity completely recovers within 45 min upon insonation. This protocol, along with the versatility of the microfluidic platform, allows to quantitatively characterise cavitation-induced events for its potential employment in clinics

Design, fabrication, and characterization of a multimodal reconfigurable bioreactor for bone tissue engineering

In the past decades, bone tissue engineering developed and exploited many typologies of bioreactors, which, besides providing proper culture conditions, aimed at integrating those bio-physical stimulations that cells experience in vivo, to promote osteogenic differentiation. Nevertheless, the highly challenging combination and deployment of many stimulation systems into a single bioreactor led to the generation of several unimodal bioreactors, investigating one or at mostly two of the required biophysical stimuli. These systems miss the physiological mimicry of bone cells environment, and often produced contrasting results, thus making the knowledge of bone mechanotransduction fragmented and often inconsistent. To overcome this issue, in this study we developed a perfusion and electroactive-vibrational reconfigurable stimulation bioreactor to investigate the differentiation of SaOS-2 bone-derived cells, hosting a piezoelectric nanocomposite membrane as cell culture substrate. This multimodal perfusion bioreactor is designed based on a numerical (finite element) model aimed at assessing the possibility to induce membrane nano-scaled vibrations (with ~12 nm amplitude at a frequency of 939 kHz) during perfusion (featuring 1.46 dyn cm−2 wall shear stress), large enough for inducing a physiologically-relevant electric output (in the order of 10 mV on average) on the membrane surface. This study explored the effects of different stimuli individually, enabling to switch on one stimulation at a time, and then to combine them to induce a faster bone matrix deposition rate. Biological results demonstrate that the multimodal configuration is the most effective in inducing SaOS-2 cell differentiation, leading to 20-fold higher collagen deposition compared to static cultures, and to 1.6- and 1.2-fold higher deposition than the perfused- or vibrated-only cultures. These promising results can provide tissue engineering scientists with a comprehensive and biomimetic stimulation platform for a better understanding of mechanotransduction phenomena beyond cells differentiation

Reversible Cavitation-Induced Junctional Opening in an Artificial Endothelial Layer.

Targeting pharmaceuticals through the endothelial barrier is crucial for drug delivery. In this context, cavitation-assisted permeation shows promise for effective and reversible opening of intercellular junctions. A vessel-on-a-chip is exploited to investigate and quantify the effect of ultrasound-excited microbubbles-stable cavitation-on endothelial integrity. In the vessel-on-a-chip, the endothelial cells form a complete lumen under physiological shear stress, resulting in intercellular junctions that exhibit barrier functionality. Immunofluorescence microscopy is exploited to monitor vascular integrity following vascular endothelial cadherin staining. It is shown that microbubbles amplify the ultrasound effect, leading to the formation of interendothelial gaps that cause barrier permeabilization. The total gap area significantly increases with pressure amplitude compared to the control. Gap opening is fully reversible with gap area distribution returning to the control levels 45 min after insonication. The proposed integrated platform allows for precise and repeatable in vitro measurements of cavitation-enhanced endothelium permeability and shows potential for validating irradiation protocols for in vivo applications

Role of lysines in cytochrome c – cardiolipin interaction

Cytochrome c undergoes structural variations during the apoptotic process; such changes have been related with modifications occurring in the protein when it forms a complex with cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several studies have been performed to identify the site(s) of the protein involved in the cytochrome c/cardiolipin interaction, to date the location of this hosting region(s) remains unidentified and is a matter of debate. To gain a deeper insight into the reaction mechanism, we investigate the role that the Lys72, Lys73 and Lys79 residues play in the cytochrome c/cardiolipin interaction, as these side chains appear to be critical for cytochrome c/cardiolipin recognition. The Lys72Asn, Lys73Asn, Lys79Asn, Lys72/73Asn and Lys72/73/79Asn mutants of horse heart cytochrome c were produced and characterized by circular dichroism, UV-visible and resonance Raman spectroscopies, and the effects of the mutations on the interaction of the variants with cardiolipin have been investigated. The mutants are characterized by a subpopulation with non-native axial coordination, and are less stable than the wild type protein. Furthermore, the mutants lacking Lys72 and/or Lys79 do not bind cardiolipin and those lacking Lys73, although they form a complex with the phospholipid, do not show any peroxidase activity. These observations indicate that the Lys72, Lys73 and Lys79 residues stabilize the native axial Met80-Fe(III) coordination as well as the tertiary structure of cytochrome c. Moreover, while Lys72 and Lys79 are critical for cytochrome c/cardiolipin recognition, the simultaneous presence of Lys72, Lys73 and Lys79 is necessary for peroxidase activity of cardiolipin-bound cytochrome c

Optimized 3D co-registration of ultra-low-field and high-field magnetic resonance images

The prototypes of ultra-low-field (ULF) MRI scanners developed in recent years represent new, innovative, cost-effective and safer systems, which are suitable to be integrated in multi-modal (Magnetoencephalography and MRI) devices. Integrated ULF-MRI and MEG scanners could represent an ideal solution to obtain functional (MEG) and anatomical (ULF MRI) information in the same environment, without errors that may limit source reconstruction accuracy. However, the low resolution and signal-to-noise ratio (SNR) of ULF images, as well as their limited coverage, do not generally allow for the construction of an accurate individual volume conductor model suitable for MEG localization. Thus, for practical usage, a high-field (HF) MRI image is also acquired, and the HF-MRI images are co-registered to the ULF-MRI ones. We address here this issue through an optimized pipeline (SWIM-Sliding WIndow grouping supporting Mutual information). The co-registration is performed by an affine transformation, the parameters of which are estimated using Normalized Mutual Information as the cost function, and Adaptive Simulated Annealing as the minimization algorithm. The sub-voxel resolution of the ULF images is handled by a sliding-window approach applying multiple grouping strategies to down-sample HF MRI to the ULF-MRI resolution. The pipeline has been tested on phantom and real data from different ULF-MRI devices, and comparison with well-known toolboxes for fMRI analysis has been performed. Our pipeline always outperformed the fMRI toolboxes (FSL and SPM). The HF-ULF MRI co-registration obtained by means of our pipeline could lead to an effective integration of ULF MRI with MEG, with the aim of improving localization accuracy, but also to help exploit ULF MRI in tumor imaging

Progressive modular rebalancing system and visual cueing for gait rehabilitation in parkinson’s disease. A pilot, randomized, controlled trial with crossover

Introduction: The progressive modular rebalancing (PMR) system is a comprehensive rehabilitation approach derived from proprioceptive neuromuscular facilitation principles. PMR training encourages focus on trunk and proximal muscle function through direct perception, strength, and stretching exercises and emphasizes bi-articular muscle function in the improvement of gait performance. Sensory cueing, such as visual cues (VC), is one of the more established techniques for gait rehabilitation in PD. In this study, we propose PMR combined with VC for improving gait performance, balance, and trunk control during gait in patients with PD. Our assumption herein was that the effect of VC may add to improved motor performance induced by the PMR treatment. The primary aim of this study was to evaluate whether the PMR system plus VC was a more effective treatment option than standard physiotherapy in improving gait function in patients with PD. The secondary aim of the study was to evaluate the effect of this treatment on motor function severity. Design: Two-center, randomized, controlled, observer-blind, crossover study with a 4-month washout period. Participants: Forty individuals with idiopathic PD in Hoehn and Yahr stages 1–4. Intervention: Eight-week rehabilitation programs consisting of PMR plus VC (treatment A) and conventional physiotherapy (treatment B). Primary outcome measures: Spatiotemporal gait parameters, joint kinematics, and trunk kinematics. Secondary outcome measures: UPDRS-III scale scores. Results: The rehabilitation program was well-tolerated by individuals with PD and most participants showed improvements in gait variables and UPDRS-III scores with both treatments. However, patients who received PMR with VC showed better results in gait function with regard to gait performance (increased step length, gait speed, and joint kinematics), gait balance (increased step width and double support duration), and trunk control (increased trunk motion) than those receiving conventional physiotherapy. While crossover results revealed some differences in primary outcomes, only 37.5% of patients crossed over between the groups. As a result, our findings should be interpreted cautiously. Conclusions: The PMR plus VC program could be used to improve gait function and severity motor of motor deficit in individuals with PD