Low-intensity Pulsed Ultrasound System Research Articles

Development of a wireless ultrasonic brain stimulation system for concurrent bilateral neuromodulation in freely moving rodents.

Bilateral brain stimulation is an important modality used to investigate brain circuits and treat neurological conditions. Recently, low-intensity pulsed ultrasound (LIPUS) received significant attention as a novel non-invasive neurostimulation technique with high spatial specificity. Despite the growing interest, the typical ultrasound brain stimulation study, especially for small animals, is limited to a single target of sonication. The constraint is associated with the complexity and the cost of the hardware system required to achieve multi-regional sonication. This work presented the development of a low-cost LIPUS system with a pair of single-element ultrasound transducers to address the above problem. The system was built with a multicore processor with an RF amplifier circuit. In addition, LIPUS device was incorporated with a wireless module (bluetooth low energy) and powered by a single 3.7 V battery. As a result, we achieved an ultrasound transmission with a central frequency of 380 kHz and a peak-to-peak pressure of 480 kPa from each ultrasound transducer. The developed system was further applied to anesthetized rats to investigate the difference between uni- and bilateral stimulation. A significant difference in cortical power density extracted from electroencephalogram signals was observed between uni- and bilateral LIPUS stimulation. The developed device provides an affordable solution to investigate the effects of LIPUS on functional interhemispheric connection.

Effect of Low-Intensity Pulsed Ultrasound on the Graft-Bone Healing of Artificial Ligaments: An In Vitro and In Vivo Study

Background: As many researchers have focused on promoting the graft-bone healing of artificial ligaments, even with numerous chemical coatings, identifying a biosafe, effective, and immediately usable method is still important clinically. Purpose: (1) To determine whether a low-intensity pulsed ultrasound system (LIPUS) promotes in vitro cell viability and osteogenic differentiation and (2) to assess the applicability and effectiveness of LIPUS in promoting the graft-bone healing of artificial ligaments in vivo. Study Design: Controlled laboratory study. Methods: Polyethylene terephthalate (PET) sheets and grafts were randomly assigned to control and LIPUS groups. MC3T3-E1 preosteoblasts were cultured on PET sheets. Cell viability and morphology were evaluated using a live/dead viability assay and scanning electron microscopy. Alkaline phosphatase activity, calcium nodule formation, and Western blot were evaluated for osteogenic differentiation. For in vivo experiments, the effect of LIPUS was evaluated via an extra-articular graft-bone healing model in 48 rabbits: the osteointegration and new bone formation were tested by micro–computed tomography and histological staining, and the graft-bone bonding was tested by biomechanical testing. Results: Cell viability was significantly higher in the LIPUS group as compared with control (living and dead compared between control and LIPUS groups, P = .0489 vs P = .0489). Better adherence of cells and greater development of extracellular matrix were observed in the LIPUS group. Furthermore, LIPUS promoted alkaline phosphatase activity, calcium nodule formation, and the protein expression of collagen 1 (P = .0002) and osteocalcin (P = .0006) in vitro. Micro–computed tomography revealed higher surrounding bone mass at 4 weeks and newly formed bone mass at 8 weeks in the LIPUS group (P = .0014 and P = .0018). Histological analysis showed a narrower interface and direct graft-bone contact in the LIPUS group; the surrounding bone area at 4 weeks and the mass of newly formed bone at 4 and 8 weeks in the LIPUS group were also significantly higher as compared with control (surrounding bone, P < .0001; newly formed bone, P = .0016 at 4 weeks and P = .005 at 8 weeks). The ultimate failure load in the LIPUS group was significantly higher than in the control group (P < .0001 at 4 weeks; P = .0008 at 8 weeks). Conclusion: LIPUS promoted the viability and osteogenic differentiation of MC3T3-E1 preosteoblasts in vitro and enhanced the graft-bone healing of PET artificial ligament in vivo. Clinical Relevance: LIPUS is an effective physical stimulation to enhance graft-bone healing after artificial ligament implantation.

Comparison Between Clinically Available Low-Intensity Pulsed Ultrasound (LIPUS) and a Novel Bimodal Acoustic Signal System for Accelerating Fracture Healing.

Low-intensity pulsed ultrasound (LIPUS) accelerates fracture healing by stimulating the production of bone callus and the mineralization process. This study compared a novel bimodal acoustic signal (BMAS) device for bone fracture healing to a clinical LIPUS system (EXOGEN; Bioventus, Durham, NC, USA). Thirty rabbits underwent a bilateral fibular osteotomy. Each rabbits' legs were randomized to receive 20-min treatment daily for 18 days with BMAS or LIPUS. The latter utilizes a longitudinal ultrasonic mode only, while the former employs ultrasound-induced shear stress to promote bone formation. Power Doppler imaging (PDI) was acquired days 0, 2, 4, 7, 11, 14, and 18 post-surgery to monitor treatment response and quantified off-line. X-rays were acquired to evaluate fractures on days 0, 14, 18, and 21. Seventeen rabbits completed the study and were euthanized day 21 post-surgery. The fibulae were analyzed to determine maximum torque, initial torsional stiffness, and angular displacement at failure. ANOVAs and paired t-tests were used to compare pair-wise outcome variables for the two treatment modes on a per rabbit basis. The BMAS system induced better fracture healing with greater stiffness (BMAS 0.21 ± 0.19 versus LIPUS 0.16 ± 0.19 [Formula: see text]cm/°, p = 0.050 ) and maximum torque (BMAS 7.84 ± 5.55 versus LIPUS 6.26 ± 3.46 [Formula: see text]cm, p = 0.022 ) than the LIPUS system. Quantitative PDI assessments showed a higher amount of vascularity with LIPUS than BMAS on days 4 and 18 ( ). In conclusion, the novel BMAS technique achieved better bone fracture healing response than the current Food and Drug Administration (FDA)-approved LIPUS system.

LIPUS far-field exposimetry system for uniform stimulation of tissues in-vitro: development and validation with bovine intervertebral disc cells

Therapeutic Low-intensity Pulsed Ultrasound (LIPUS) has been applied clinically for bone fracture healing and has been shown to stimulate extracellular matrix (ECM) metabolism in numerous soft tissues including intervertebral disc (IVD). In-vitro LIPUS testing systems have been developed and typically include polystyrene cell culture plates (CCP) placed directly on top of the ultrasound transducer in the acoustic near-field (NF). This configuration introduces several undesirable acoustic artifacts, making the establishment of dose-response relationships difficult, and is not relevant for targeting deep tissues such as the IVD, which may require far-field (FF) exposure from low frequency sources. The objective of this study was to design and validate an in-vitro LIPUS system for stimulating ECM synthesis in IVD-cells while mimicking attributes of a deep delivery system by delivering uniform, FF acoustic energy while minimizing reflections and standing waves within target wells, and unwanted temperature elevation within target samples. Acoustic field simulations and hydrophone measurements demonstrated that by directing LIPUS energy at 0.5, 1.0, or 1.5 MHz operating frequency, with an acoustic standoff in the FF (125–350 mm), at 6-well CCP targets including an alginate ring spacer, uniform intensity distributions can be delivered. A custom FF LIPUS system was fabricated and demonstrated reduced acoustic intensity field heterogeneity within CCP-wells by up to 93% compared to common NF configurations. When bovine IVD cells were exposed to LIPUS (1.5 MHz, 200 μs pulse, 1 kHz pulse frequency, and ISPTA = 120 mW cm−2) using the FF system, sample heating was minimal (+0.81 °C) and collagen content was increased by 2.6-fold compared to the control and was equivalent to BMP-7 growth factor treatment. The results of this study demonstrate that FF LIPUS exposure increases collagen content in IVD cells and suggest that LIPUS is a potential noninvasive therapeutic for stimulating repair of tissues deep within the body such as the IVD.

Biophysical stimulation of bone and cartilage: state of the art and future perspectives.

IntroductionBiophysical stimulation is a non-invasive therapy used in orthopaedic practice to increase and enhance reparative and anabolic activities of tissue.MethodsA sistematic web-based search for papers was conducted using the following titles: (1) pulsed electromagnetic field (PEMF), capacitively coupled electrical field (CCEF), low intensity pulsed ultrasound system (LIPUS) and biophysical stimulation; (2) bone cells, bone tissue, fracture, non-union, prosthesis and vertebral fracture; and (3) chondrocyte, synoviocytes, joint chondroprotection, arthroscopy and knee arthroplasty.ResultsPre-clinical studies have shown that the site of interaction of biophysical stimuli is the cell membrane. Its effect on bone tissue is to increase proliferation, synthesis and release of growth factors. On articular cells, it creates a strong A2A and A3 adenosine-agonist effect inducing an anti-inflammatory and chondroprotective result. In treated animals, it has been shown that the mineralisation rate of newly formed bone is almost doubled, the progression of the osteoarthritic cartilage degeneration is inhibited and quality of cartilage is preserved. Biophysical stimulation has been used in the clinical setting to promote the healing of fractures and non-unions. It has been successfully used on joint pathologies for its beneficial effect on improving function in early OA and after knee surgery to limit the inflammation of periarticular tissues.DiscussionThe pooled result of the studies in this review revealed the efficacy of biophysical stimulation for bone healing and joint chondroprotection based on proven methodological quality.ConclusionThe orthopaedic community has played a central role in the development and understanding of the importance of the physical stimuli. Biophysical stimulation requires care and precision in use if it is to ensure the success expected of it by physicians and patients.

Low intensity pulsed ultrasound (LIPUS) for the treatment of intervertebral disc degeneration.

Discogenic back pain presents a major public health issue, with current therapeutic interventions limited to short-term symptom relief without providing regenerative remedies for diseased intervertebral discs (IVD). Many of these interventions are invasive and can diminish the biomechanical integrity of the IVDs. Low intensity pulsed ultrasound (LIPUS) is a potential treatment option that is both non-invasive and regenerative. LIPUS has been shown to be a clinically effective method for the enhancement of wound and fracture healing. Recent in vitro studies have shown that LIPUS stimulation induces an upregulation functional matrix proteins and downregulation of inflammatory factors in cultured IVD cells. However, we do not know the effects of LIPUS on an in vivo model for intervertebral disc degeneration. The objective of this study was to show technical feasibility of building a LIPUS system that can target the rat tail IVD and apply this setup to a model for acute IVD degeneration. A LIPUS exposimetry system was built using a 1.0 MHz planar transducer and custom housing. Ex vivo intensity measurements demonstrated LIPUS delivery to the center of the rat tail IVD. Using an established stab-incision model for disc degeneration, LIPUS was applied for 20 minutes daily for five days. For rats that displayed a significant injury response, LIPUS treatment caused significant upregulation of Collagen II and downregulation of Tumor Necrosis Factor - α gene expression. Our preliminary studies indicate technical feasibility of targeted delivery of ultrasound to a rat tail IVD for studies of LIPUS biological effects.

Applications of Ultrasound to Enhance Mycophenolic Acid Production

Reducing production costs for fermentation-based drugs (e.g., antibiotics) is crucial for the long-term sustainability of healthcare. In this study, we propose a novel low-intensity pulsed ultrasound (LIPUS) stimulation scheme using a nominal frequency of 1.5 MHz with a 20% duty cycle (200 μs ultrasound on and 800 μs ultrasound off) to increase production of fermentation-based drugs. We chose Penicillium brevicompactum as a model system to demonstrate the performance of our LIPUS system. Penicillium brevicompactum can produce mycophenolic acid (MPA), an immunosuppressive agent commonly used to prevent rejection after organ transplantation. We have stimulated Penicillium brevicompactum in 50 mL shake flasks using LIPUS during its fermentation. After a series of screening experiments to optimize ultrasound parameters (e.g., ultrasound intensities, treatment duration and treatment frequency per day), it was concluded that ultrasound stimulation can increase MPA production by as much as 60% when treated eight times a day for 10-min durations at an intensity (spatial peak temporal average) of 200 mW/cm2. These findings elucidate a new approach to reduce the cost of producing fermentation-based drugs.

The development and validation of a lipus system with preliminary observations of ultrasonic effects on human adult stem cells

To study the potential effects of low-intensity pulsed ultrasound (LIPUS) on cell response in vitro, the ability to alter LIPUS parameters is required. However, commercial LIPUS systems have very little control over parameter selection. In this study, a custom LIPUS system was designed and validated by exploring the effects of using different pulse repetition frequency (PRF) parameters on human adipose derived adult stem cells (hASCs) and bone marrow derived mesenchymal stem cells (hMSCs), two common stem cell sources for creating bone constructs in vitro. Changing the PRF was found to affect cellular response to LIPUS stimulation for both cell types. Proliferation of LIPUS-stimulated cells was found to decrease for hASCs by d 7 for all three groups compared with unstimulated control cells (P = 0.008, 0.011, 0.014 for 1 Hz, 100 Hz and 1 kHz PRF, respectively) and for hMSCs by d 14 (donor 1: P = 0.0005, 0.0002, 0.0003; donor 2: P = 0.0003, 0.0002, 0.0001; for PRFs of 1 Hz, 100 Hz, and 1 kHz, respectively). Additionally, LIPUS was shown to strongly accelerate osteogenic differentiation of hASCs based on amount of calcium accretion normalized by total DNA (P = 0.003, 0.001, 0.003, and 0.032 between control/100 Hz, control/1 kHz, 1 Hz/1 kHz, and 100 Hz/1 kHz pulse repetition frequencies, respectively). These findings promote the study of using LIPUS to induce osteogenic differentiation and further encourage the exploration of LIPUS parameter optimization. The custom LIPUS system was successfully designed to allow extreme parameter variation, specifically PRF, and encourages further studies.