Ultrasound Stimulation of Piezoelectric Nanocomposite Hydrogels Boosts Chondrogenic Differentiation in Vitro, in Both a Normal and Inflammatory Milieu.

The use of piezoelectric nanomaterials combined with ultrasound stimulation is emerging as a promising approach for wirelessly triggering the regeneration of different tissue types. However, it has never been explored for boosting chondrogenesis. Furthermore, the ultrasound stimulation parameters used are often not adequately controlled. In this study, we show that adipose-tissue-derived mesenchymal stromal cells embedded in a nanocomposite hydrogel containing piezoelectric barium titanate nanoparticles and graphene oxide nanoflakes and stimulated with ultrasound waves with precisely controlled parameters (1 MHz and 250 mW/cm2, for 5 min once every 2 days for 10 days) dramatically boost chondrogenic cell commitment in vitro. Moreover, fibrotic and catabolic factors are strongly down-modulated: proteomic analyses reveal that such stimulation influences biological processes involved in cytoskeleton and extracellular matrix organization, collagen fibril organization, and metabolic processes. The optimal stimulation regimen also has a considerable anti-inflammatory effect and keeps its ability to boost chondrogenesis in vitro, even in an inflammatory milieu. An analytical model to predict the voltage generated by piezoelectric nanoparticles invested by ultrasound waves is proposed, together with a computational tool that takes into consideration nanoparticle clustering within the cell vacuoles and predicts the electric field streamline distribution in the cell cytoplasm. The proposed nanocomposite hydrogel shows good injectability and adhesion to the cartilage tissue ex vivo, as well as excellent biocompatibility in vivo, according to ISO 10993. Future perspectives will involve preclinical testing of this paradigm for cartilage regeneration.

Low-intensity pulsed ultrasound increases neurotrophic factors secretion and suppresses inflammation inin vitromodels of peripheral neuropathies.

Objective. In this study, we aimed to verify the beneficial effects of low-intensity pulsed ultrasound (LIPUS) stimulation on two cell types: H2O2-treated RSC96 Schwann cells and THP-1 macrophages, used to model neuropathic inflammation.Approach. Using a set-up guaranteeing a fine control of the ultrasound dose at the target, different frequencies (38 kHz, 1 MHz, 5 MHz) and different intensities (20, 100, 500 mW cm-2) were screened to find the most effective experimental conditions for triggering beneficial effects on metabolic activity and release of neurotrophic cytokines (ß-nerve growth factor, brain-derived neurotrophic factor, glial cell-derived neurotrophic factor) of RSC96 cells. The combination of parameters resulting the optimal one was applied to evaluate anti-inflammatory effects in terms of reactive oxygen species (ROS) and tumor necrosis factor-α(TNF-α) production, also investigating a possible anti-oxidant activity and mechanotransduction pathway for the anti-inflammatory process. The same optimal combination of parameters was then applied to THP-1 cells, differentiated into M1 and M2 phenotypes, to assess the effect on the expression and release of pro-inflammatory markers (TNF-α, interleukin (IL)-1ß, IL-6, IL-8) and anti-inflammatory ones (IL-10 and CD206).Main results.5 MHz and 500 mW cm-2were found as the optimal stimulation parameters on RSC96 cells. Such parameters were also found to suppress ROS and TNF-αin the same cell line, thus highlighting a possible anti-inflammatory effect, involving the NF-kB pathway. An anti-oxidant effect induced by LIPUS was also observed. Finally, the same LIPUS parameters did not induce any differentiation towards the M1 phenotype of THP-1 cells, whereas they decreased TNF-αand IL-8 gene expression, reduced IL-8 cytokine release and increased IL-10 cytokine release in M1-polarized THP-1 cells.Significance.This study represents the first step towards the use of precisely controlled LIPUS for the treatment of peripheral neuropathies.

Optimal low-intensity pulsed ultrasound stimulation for promoting anti-inflammatory effects in macrophages.

In this paper, we stimulated M1-like macrophages (obtained from U937 cells) with low-intensity pulsed ultrasound (LIPUS) to lower pro-inflammatory cytokine production. A systematic screening of different frequencies, intensities, duty cycles, and exposure times was performed. The optimal stimulation conditions leading to a marked decrease in the release of inflammatory cytokines were determined to be 38 kHz, 250 mW/cm2, 20%, and 90 min, respectively. Using these parameters, we verified that up to 72 h LIPUS did not affect cell viability, resulting in an increase in metabolic activity and in a reduction of reactive oxygen species (ROS) production. Moreover, we found that two mechanosensitive ion channels (PIEZO1 and TRPV1) were involved in the LIPUS-mediated cytokine release modulation. We also assessed the role of the nuclear factor κB (NF-κB) signaling pathway and observed an enhancement of actin polymerization. Finally, transcriptomic data suggested that the bioeffects of LIPUS treatment occur through the modulation of p38 MAPK signaling pathway.

Piezoelectric Nanomaterials Activated by Ultrasound: The Pathway from Discovery to Future Clinical Adoption.

Electrical stimulation has shown great promise in biomedical applications, such as regenerative medicine, neuromodulation, and cancer treatment. Yet, the use of electrical end effectors such as electrodes requires connectors and batteries, which dramatically hamper the translation of electrical stimulation technologies in several scenarios. Piezoelectric nanomaterials can overcome the limitations of current electrical stimulation procedures as they can be wirelessly activated by external energy sources such as ultrasound. Wireless electrical stimulation mediated by piezoelectric nanoarchitectures constitutes an innovative paradigm enabling the induction of electrical cues within the body in a localized, wireless, and minimally invasive fashion. In this review, we highlight the fundamental mechanisms of acoustically mediated piezoelectric stimulation and its applications in the biomedical area. Yet, the adoption of this technology in a clinical practice is in its infancy, as several open issues, such as piezoelectric properties measurement, control of the ultrasound dose in vitro, modeling and measurement of the piezo effects, knowledge on the triggered bioeffects, therapy targeting, biocompatibility studies, and control of the ultrasound dose delivered in vivo, must be addressed. This article explores the current open challenges in piezoelectric stimulation and proposes strategies that may guide future research efforts in this field toward the translation of this technology to the clinical scene.

Acoustic Coupling Quantification in Ultrasound-Guided Focused Ultrasound Surgery: Simulation-Based Evaluation and Experimental Feasibility Study.

Adequate acoustic coupling between the therapeutic transducer and the patient's body is essential for safe and efficient focused ultrasound surgery (FUS). There is currently no quantitative method for acoustic coupling verification in ultrasound-guided FUS. In this work, a quantitative method was developed and a related metric was introduced: the acoustic coupling coefficient. This metric associates the adequacy of the acoustic coupling with the reflected signals recorded through an imaging probe during a low-energy sonication. The acoustic coupling issue was simulated in silico and validated through in vitro tests. Our results indicated a sigmoidal behavior of the introduced metric as the contact surface between the coupling system and the patient's skin increases. The proposed method could be a safety-check criterion for verifying the adequacy of the acoustic coupling before starting the FUS treatment, thus ensuring efficient energy transmission to the target and preventing damage to both the patient and the instrumentation.

Pulsatile Drug Delivery System Triggered by Acoustic Radiation Force.

Since biological systems exhibit a circadian rhythm (24-hour cycle), they are susceptible to the timing of drug administration. Indeed, several disorders require a therapy that synchronizes with the onset of symptoms. A targeted therapy with spatially and temporally precise controlled drug release can guarantee a considerable gain in terms of efficacy and safety of the treatment compared to traditional pharmacological methods, especially for chronotherapeutic disorders. This paper presents a proof of concept of an innovative pulsatile drug delivery system remotely triggered by the acoustic radiation force of ultrasound. The device consists of a case, in which a drug-loaded gel can be embedded, and a sliding top that can be moved on demand by the application of an acoustic stimulus, thus enabling drug release. Results demonstrate for the first time that ultrasound acoustic radiation force (up to 0.1 N) can be used for an efficient pulsatile drug delivery (up to 20 µg of drug released for each shot).

Ultrasound Stimulations Induce Prolonged Depolarization and Fast Action Potentials in Leech Neurons.

Objective: Ultrasound (US) stimulation carries the promise of a selective, reversible, and non-invasive modulation of neural activity without the need for genetic manipulation of neural structures. However, the mechanisms of US-induced generation of action potentials (APs) are still unclear. Methods: Here we address this issue by analyzing intracellularly recorded responses of leech nociceptive neurons to controlled delivery of US. Results: US induced a depolarization linearly accumulating in time and outlasting the duration of the stimulation. Spiking activity was reliably induced for an optimal US intensity range. Moreover, we found that APs induced by US differ in smaller amplitude and faster repolarization from those induced by electrical stimulation in the same cell but display the same repolarization rate. Conclusions: These results shed light on the mechanism by which spikes are induced by US and pave the way for designing more efficient US stimulation patterns.

Small-caliber vascular grafts based on a piezoelectric nanocomposite elastomer: Mechanical properties and biocompatibility.

The development of small-caliber grafts still represents a challenge in the field of vascular prostheses. Among other factors, the mechanical properties mismatch between natural vessels and artificial devices limits the efficacy of state-of-the-art materials. In this paper, a novel nanocomposite graft with an internal diameter of 6 mm is proposed. The device is obtained through spray deposition using a semi-interpenetrating polymeric network combining poly(ether)urethane and polydimethilsyloxane. The inclusion of BaTiO3 nanoparticles endows the scaffold with piezoelectric properties, which may be exploited in the future to trigger beneficial biological effects. Graft characterization demonstrated a good nanoparticle dispersion and an overall porosity that was not influenced by the presence of nanoparticles. Graft mechanical properties resembled (or even ameliorated) the ones of natural vessels: both doped and non-doped samples showed a Young's modulus of ∼700 kPa in the radial direction and ∼900 kPa in the longitudinal direction, an ultimate tensile strength of ∼1 MPa, a strain to failure of ∼700%, a suture retention force of ∼1.7 N and a flexural rigidity of ∼2.5 × 10-5 N m2. The two grafts differed in terms of burst strength that resulted ∼800 kPa for the control non-doped samples and ∼1100 kPa for the doped ones. The graft doped with BaTiO3 nanoparticles showed a d33 coefficient of 1.91 pm/V, almost double than the non-doped control. The device resulted highly stable, with a mass loss smaller than 2% over 3 months and an excellent biocompatibility.

Feedback control of microbubble cavitation for ultrasound-mediated blood-brain barrier disruption in non-human primates under magnetic resonance guidance.

Focused ultrasound (FUS) in combination with microbubbles is capable of noninvasive, site-targeted delivery of drugs through the blood-brain barrier (BBB). Although acoustic parameters are reproducible in small animals, their control remains challenging in primates due to skull heterogeneity. This study describes a 7-T magnetic resonance (MR)-guided FUS system designed for BBB disruption in non-human primates (NHP) with a robust feedback control based on passive cavitation detection (PCD). Contrast enhanced T1-weighted MR images confirmed the BBB opening in NHP sonicated during 2 min with 500-kHz frequency, pulse length of 10 ms, and pulse repetition frequency of 5 Hz. The safe acoustic pressure range from 185 ± 22 kPa to 266 ± 4 kPa in one representative case was estimated from combining data from the acoustic beam profile with the BBB opening and hemorrhage profiles obtained from MR images. A maximum amount of MR contrast agent at focus was observed at 30 min after sonication with a relative contrast enhancement of 67% ± 15% (in comparison to that found in muscles). The feedback control based on PCD using relative spectra was shown to be robust, allowing comparisons across animals and experimental sessions. Finally, we also demonstrated that PCD can test acoustic coupling conditions, which improves the efficacy and safety of ultrasound transmission into the brain.

Optimal Ultrasound Exposure Conditions for Maximizing C2C12 Muscle Cell Proliferation and Differentiation.

Described here is an in vitro systematic investigation of the effects on C2C12 myoblasts of exposure to finely controlled and repeatable low-intensity pulsed ultrasound of different frequencies (500 kHz, 1 MHz, 3 MHz and 5 MHz) and different intensities (250, 500 and 1000 mW/cm2). An in-house stimulation system and an ultrasound-transparent cell culture well minimized reflections and attenuations, allowing precise control of ultrasound delivery. Results indicated that a 3 MHz stimulation at 1 W/cm2 intensity maximized cell proliferation in comparison with the other exposure conditions and untreated controls. In contrast, cell differentiation and the consequent formation of multinucleated myotubes were maximized by 1 MHz stimulation at 500 mW/cm2 intensity. The highly controlled exposure conditions employed allowed precise correlation of the ultrasound delivery to the bio-effects produced, thus overcoming the inconsistency of some results available in the literature and contributing to the potential of ultrasound treatment for muscle therapy and regeneration.