Ultrasound is considered a safe and non-invasive tool in regenerative medicine. In particular, low-intensity pulsed ultrasound (LIPUS) has been used in the clinic for more than twenty years for applications in bone healing. It has been demonstrated to be an effective tool to treat different chronic diseases. We sought to evaluate the effects produced by LIPUS on the properties of human breast cancer stem cells (bCSCs).

Cells were stimulated using a traditional ultrasound device with the following parameters: 0.05 W/cm2 with 10% duty cycle, frequency of 3 MHz and 8 pulses.

At the parameters used, the ultrasound did not directly affect bCSC proliferation, with no evident changes in morphology. In contrast, the ultrasound protocol affected the migration and invasion ability of bCSCs, limiting their capacity to advance while a major affection was detected on their angiogenic properties. LIPUS-treated bCSCs were unable to transform into aggressive metastatic cancer cells, by decreasing their migration and invasion capacity as well as vessel formation. Finally, RNA-seq analysis revealed major changes in gene expression, with 676 differentially expressed genes after LIPUS stimulation, 578 upregulated and 98 downregulated.

Overall, these results highlight the potential of LIPUS as a promising non-invasive therapy to target bCSCs and attenuate its capacity to drive migration, invasion, angiogenesis and, ultimately, tumor malignancy. Besides, the ability of LIPUS to modulate gene expression points out its capacity to broadly influence the cellular transcriptome. Therefore, the application of LIPUS as an antitumor therapeutic agent targeting bCSCs may offer a promising new approach to treat cancer. In vivo functional experiments will determine in the future the relevance of LIPUS application for the development of metastasis.

Addressing the concurrent repair of cartilage and subchondral bone presents a significant challenge yet is crucial for the effective treatment of severe joint injuries. This study introduces a novel biodegradable composite scaffold, integrating piezoelectric poly-l-lactic acid (pPLLA) with strontium-enriched silicate bioceramic (SrSiO3). This innovative scaffold continually releases bioactive Sr2+ and SiO32- ions while generating an electrical charge under low-intensity pulsed ultrasound (LIPUS) stimulation, a clinically recognized method. The scaffold's unique dual action, emanating both chemical and electrical signals, activates the purinergic receptor P2X 1 (P2RX1) calcium ion channel, promoting an influx of intracellular calcium ions. This process results in a synergistic enhancement of both chondrogenic activities of rat chondrocytes (rCCs) and osteogenic activities of rat bone marrow mesenchymal stem cells (rBMSCs). Furthermore, the scaffold's effectiveness in integrating articular cartilage and subchondral bone repair is confirmed in a rat model of joint osteochondral injury. This study thereby offers a groundbreaking approach for treating severe osteoarticular cartilage defects.

Endoplasmic reticulum (ER) stress is associated with oxidative stress, which is integral to the development of various pathological conditions, including neurodegenerative disorders. In this study, using NSC-34-a hybrid cell line established by fusing motor neuron-rich embryonic spinal cord cells with mouse neuroblastoma cells-we investigated the effects of low-intensity pulsed ultrasound (LIPUS) stimulation on oxidative (reactive oxygen species)/ER stress-induced neurodegeneration. An ultrasound transducer with a center frequency of 1.15 MHz and a spatial peak temporal average intensity of 357 mW/cm2 was used for delivering ultrasound (for 8 min, via a water-filled tube) to motor neuron cells seeded in a plastic culture dish. LIPUS stimulation significantly increased the level of the antiapoptotic protein B-cell lymphoma 2 (BCL-2) and inhibited the expression of apoptosis-associated proteins such as BCL-2-associated X protein (BAX), CCAAT/enhancer-binding protein-homologous protein (CHOP), and caspase-12, thus extending the survival of motor neurons. LIPUS stimulation also enhanced Ca2+ signaling and activated the Ca2+-dependent transcription factors as nuclear factor of activated T cells (NFAT) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Furthermore, LIPUS stimulation induced the activation of the serine/threonine kinase protein kinase B (AKT). Thus, LIPUS stimulation prevented oxidative/ER stress-mediated mitochondrial dysfunction. In conclusion, as a safe and noninvasive method, LIPUS stimulation can facilitate further development of ultrasound neuromodulation as a tool for neuroscience research.

Acute Kidney Injury (AKI) is a significant medical condition characterized by the abrupt decline in kidney function.Low-intensity pulsed ultrasound (LIPUS), a non-invasive therapeutic technique employing low-intensity acoustic wave pulses, has shown promise in promoting tissue repair and regeneration. A novel LIPUS system was developed and evaluated in rat AKI models, focusing on its effects on glomerular filtration rate (GFR), blood urea nitrogen (BUN), serum creatinine (SCr), and the Notch1-Akt-eNOS signaling pathway. The results demonstrated that LIPUS treatment improved GFR, BUN, SCr levels, and renal pathology in AKI rats. In vitro experiments using HUVEC cells revealed that LIPUS stimulation promoted angiogenesis, cell migration mechanically-dependent calcium ion influx, which was partially attenuated by TRPV1 knockdown. RNA sequencing analysis indicated LIPUS-induced activation of the Notch pathway, phosphorylation of Akt and eNOS. Furthermore, inhibition or genetic silencing of Notch1 abolished the beneficial effects of LIPUS on angiogenesis, renal function, and Akt-eNOS phosphorylation in both cells and AKI rats. These findings suggest that LIPUS-induced calcium influx promotes Akt-eNOS phosphorylation, nitric oxide (NO) production, angiogenesis, and improved renal function in AKI via Notch1-Akt-eNOS signaling, positioning LIPUS as a promising therapeutic strategy for AKI by targeting vascular regeneration.

Vascularization is a major challenge in the field of tissue engineering and regenerative medicine. Mechanical factors have been demonstrated to play a fundamental role in vasculogenesis and angiogenesis and can affect the architecture of the generated vascular network. Through the regulation of mechanical factors in engineered tissues, various mechanical strategies can be used to optimize the preformed vascular network and promote its rapid integration with host vessels. Optimization of the mechanical properties of scaffolds, including controlling scaffold stiffness, increasing surface roughness and anisotropic structure, and designing interconnected, hierarchical pore structures, is beneficial for the in vitro formation of vascular networks and the ingrowth of host blood vessels. The incorporation of hollow channels into scaffolds promotes the formation of patterned vascular networks. Dynamic stretching and perfusion can facilitate the formation and maturation of preformed vascular networks in vitro. Several indirect mechanical strategies provide sustained mechanical stimulation to engineered tissues in vivo, which further promotes the vascularization of implants within the body. Additionally, stiffness gradients, anisotropic substrates and hollow channels in scaffolds, as well as external cyclic stretch, boundary constraints and dynamic flow culture, can effectively regulate the alignment of vascular networks, thereby promoting better integration of prevascularized engineered tissues with host blood vessels. This review summarizes the influence and contribution of both scaffold-based and external stimulus-based mechanical strategies for vascularization in tissue engineering and elucidates the underlying mechanisms involved.

Mesenchymal stem cells (MSCs) are naturally-derived regenerative materials that exhibit significant potential in regenerative medicine. Previous studies have demonstrated that MSCs-based therapy can improve heart function in ischemia-injured hearts, offering an exciting therapeutic intervention for myocardial ischemic infarction, a leading cause of worldwide mortality and disability. However, the efficacy of MSCs-based therapies is significantly disturbed by the myocardial microenvironment, which undergoes substantial changes following ischemic injury. After the ischemic injury, blood vessels become obstructed and damaged, and cardiomyocytes experience ischemic conditions. This activates the hypoxia-induced factor 1 (HIF-1) pathway, leading to the rapid production of several cytokines and chemokines, including vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (SDF-1), which are crucial for angiogenesis, cell migration, and tissue repair, but it is not sustainable. MSCs respond to these cytokines and chemokines by homing to the injured site and participating in myocardial regeneration. However, the deteriorated microenvironment in the injured myocardium poses challenges for cell survival, interacting with MSCs, and constraining their homing, retention, and migration capabilities, thereby limiting their regenerative potential. This review discusses how the deteriorated microenvironment negatively affects the ability of MSCs to promote myocardial regeneration. Recent studies have shown that optimizing the microenvironment through the promotion of angiogenesis can significantly enhance the efficacy of MSCs in treating myocardial infarction. This approach harnesses the full therapeutic potential of MSCs-based therapies for ischemic heart disease.

The periosteum plays a vital role in repairing bone defects. Researchers have demonstrated the existence of electrical potential in the periosteum and native bone, indicating that electrical signals are essential for functional bone regeneration. However, the clinical use of external electrical treatments has been limited due to their inconvenience and inefficacy. As an alternative, low-intensity pulsed ultrasound (LIPUS) is a noninvasive form of physical therapy that enhances bone regeneration. Furthermore, the wireless activation of piezoelectric biomaterials through ultrasound stimulation would generate electric charges precisely at the defect area, compensating for the insufficiency of external electrical stimulation and potentially promoting bone regeneration through the synergistic effect of mechanical and electrical stimulation. However, the optimal integration of LIPUS with an appropriate piezoelectric periosteum is yet to be explored. Herein, the BaTiO3/multiwalled-carbon nanotubes/collagen (BMC) membranes have been fabricated, possessing physicochemical properties including improved surface hydrophilicity, enhanced mechanical performance, ideal piezoelectricity, and outstanding biocompatibility, all of which are conducive to bone regeneration. When combined with LIPUS, the endogenous electrical microenvironment of native bone was recreated. After that, the wireless-generated electrical signals, along with the mechanical signals induced by LIPUS, were transferred to macrophages and activated Ca2+ influx through Piezo1. Ultimately, the regenerative effect of the BMC membrane with LIPUS stimulation (BMC + L) was confirmed in a mouse cranial defect model. Together, this research presents a co-engineering strategy that involves fabricating a novel biomimetic periosteum and utilizing the synergistic effect of ultrasound to enhance bone regeneration, which is achieved through the reinforcement of the electrical environment and the immunomodulation of macrophage polarization.

Low-Intensity Pulsed Ultrasound (LIPUS) holds therapeutic potential in promoting skeletal muscle regeneration, a biological process mediated by satellite cells and myoblasts. Despite their central roles in regeneration, the detailed mechanistic of LIPUS influence on satellite cells and myoblasts are not fully underexplored. In the current investigation, we administrated LIPUS treatment to injured skeletal muscles and C2C12 myoblasts over five consecutive days. Muscle samples were collected on days 6 and 30 post-injury for an in-depth histological and molecular assessment, both in vivo and in vitro with immunofluorescence analysis. During the acute injury phase, LIPUS treatment significantly augmented the satellite cell population, concurrently enhancing the number and size of newly formed myofibers whilst reducing fibrosis levels. At 30 days post-injury, the LIPUS-treated group demonstrated a more robust satellite cell pool and a higher myofiber count, suggesting that early LIPUS intervention facilitates satellite cell proliferation and differentiation, thereby promoting long-term recovery. Additionally, LIPUS markedly accelerated C2C12 myoblast differentiation, with observed increases in AMPK phosphorylation in myoblasts, leading to elevated expression of Glut4 and PGC-1α, and subsequent glucose uptake and mitochondrial biogenesis. These findings imply that LIPUS-induced modulation of myoblasts may culminate in enhanced cellular energy availability, laying a theoretical groundwork for employing LIPUS in ameliorating skeletal muscle regeneration post-injury. NEW & NOTEWORTHY: Utilizing the cardiotoxin (CTX) muscle injury model, we investigated the influence of LIPUS on satellite cell homeostasis and skeletal muscle regeneration. Our findings indicate that LIPUS promotes satellite cell proliferation and differentiation, thereby facilitating skeletal muscle repair. Additionally, in vitro investigations lend credence to the hypothesis that the regulatory effect of LIPUS on satellite cells may be attributed to its capability to enhance cellular energy metabolism.

Tendon injuries are the most common soft tissue injuries, caused by tissue overuse and age-related degeneration. However, the tendon repair process is slow and inefficient due to the lack of cellular structure and blood vessels in the tendon. Low-intensity pulsed ultrasound (LIPUS) has received increasing attention as a non-invasive, simple, and safe way to promote tendon healing. This review summarizes the effects and underlying mechanisms of LIPUS on tendon injury by comprehensively examining the published literature, including in vitro, in vivo, and clinical studies. This review reviewed 24 studies, with 87.5% showing improvement. The application of LIPUS in tendon diseases is a promising field worthy of further study.

All cells possess an innate ability to respond to a range of mechanical stimuli through their complex internal machinery. This comprises various mechanosensory elements that detect these mechanical cues and diverse cytoskeletal structures that transmit the force to different parts of the cell, where they are transcribed into complex transcriptomic and signaling events that determine their response and fate. In contrast to static (or steady) mechanostimuli primarily involving constant-force loading such as compression, tension, and shear (or forces applied at very low oscillatory frequencies (≤ 1 Hz) that essentially render their effects quasi-static), dynamic mechanostimuli comprising more complex vibrational forms (e.g., time-dependent, i.e., periodic, forcing) at higher frequencies are less well understood in comparison. We review the mechanotransductive processes associated with such acoustic forcing, typically at ultrasonic frequencies (> 20 kHz), and discuss the various applications that arise from the cellular responses that are generated, particularly for regenerative therapeutics, such as exosome biogenesis, stem cell differentiation, and endothelial barrier modulation. Finally, we offer perspectives on the possible existence of a universal mechanism that is common across all forms of acoustically driven mechanostimuli that underscores the central role of the cell membrane as the key effector, and calcium as the dominant second messenger, in the mechanotransduction process.

Our previous study demonstrated that ultrasound is able to promote differentiation on neural stem cells (NSCs), and dual-frequency ultrasound promotes this effect due to enhanced acoustic cavitation compared with single-frequency ultrasound. However, the underlying biological reasons have not been well disclosed. The purpose of this study was to investigate the underlying bioeffects, mechanisms and signaling pathways of dual-frequency ultrasound on NSC differentiation. The morphology, neurite outgrowth, and differentiation percentages were investigated under various dual-frequency simulation parameters with exposure periods varying from 5 to 15 min. Morphological observations identified that dual-frequency ultrasound stimulation promoted ultrasound dose-dependent neurite outgrowth. In particular, cells exposed for 10 min/2 days showed optimal neurite outgrowth and neuron differentiation percentages. In addition, live cell calcium images showed that dual-frequency ultrasound enhanced the internal calcium content of the cells, and calcium ions entering cells from the extracellular environment could be observed. Dual frequency ultrasound exposure enhanced extracellular calcium influx and upregulated extracellular signal-regulated kinases 1/2 (ERK1/2) expression. Observations from immunostaining and protein expression examinations also identified that dual-frequency ultrasound promoted brain-derived neurotrophic factor (BDNF) secretion from astrocytes derived from NSCs. In summary, evidence supports that dual-frequency ultrasound effectively enhances functional neuron differentiation via calcium channel regulation via the downstream ERK1/2 pathway and promotes BDNF secretion to serve as feedback to cascade neuron differentiation. The results may provide an alternative for cell-based therapy in brain injury.

Motor neuron diseases (MND) including amyotrophic lateral sclerosis and Parkinson disease are commonly neurodegenerative, causing a gradual loss of nerve cells and affecting the mechanisms underlying changes in calcium (Ca²⁺)-regulated dendritic growth. In this study, the NSC-34 cell line, a population of hybridomas generated using mouse spinal cord cells with neuroblastoma, was used to investigate the effect of low-intensity pulsed ultrasound (LIPUS) as part of an MND treatment model. After NSC-34 cells were seeded for 24 h, LIPUS stimulation was performed on the cells at days 1 and 3 using a non-focused transducer at 1.15 MHz for 8 min. NSC-34 cell proliferation and morphological changes were observed at various LIPUS intensities and different combinations of Ca²⁺ channel blockers. The nuclear translocation of Ca²⁺-dependent transcription factors was also examined. We observed that the neurite outgrowth and cell number of NSC-34 significantly increased with LIPUS stimulation at days 2 and 4, which may be associated with the treatment's positive effect on the activation of Ca²⁺-dependent transcription factors, such as nuclear factor of activated T cells and nuclear factor-kappa B. Our findings suggest that the LIPUS-induced Ca²⁺ signaling and transcription factor activation facilitate the morphological maturation and proliferation of NSC-34 cells, presenting a promising noninvasive method to improve stimulation therapy for MNDs in the future.

Recently, we read with interest the article entitled "Unveiling the Morphogenetic Code: A New Path at the Intersection of Physical Energies and Chemical Signaling". In this paper, the investigation into the systematic and comprehensive bio-effects of physical energies prompted us to reflect on our research. We believe that ultrasound, which possesses a special physical energy, also has a certain positive regulatory effect on macrophages, and we have already obtained some preliminary research results that support our hypothesis.