Soft tissue stabilization of the hinge position in medial closed wedge distal femoral osteotomy: an anatomical study.

BACKGROUND: Soft tissue has an important role in stabilizing the hinge point of medial closed wedge distal femoral osteotomy (MCWDFO). However, there are conflicting data on the soft tissue anatomy around the hinge point of MCWDFO and, therefore, further anatomical data are needed. The purposes of the study were to: 1) anatomically analyze the soft tissue around the hinge point of MCWDFO; 2) radiologically define the appropriate hinge point to prevent an unstable hinge fracture based on the result of the anatomical analysis; and 3) histologically analyze the soft tissue based on the result of the anatomical analysis. METHODS: In 20 cadaveric knees, the capsule attachment of the distal lateral side of the femur was marked with a radiopaque ball bearing. A digital planning tool was used to calculate the area of the marked capsule attachment around the ideal hinge point of MCWDFO on radiographs. The soft tissue around the hinge point was histologically examined and the periosteal thickness was measured and visualized graphically. The graph and radiograph were overlayed using image editing software, and the appropriate hinge position was determined based on the periosteal thickness. RESULTS: As a result, the periosteal thickness of the distal lateral femur tended to rapidly decrease from the metaphyseal region toward the diaphyseal region. The overlayed graph and radiograph revealed that the periosteal thickness changed in the region corresponding to the apex of the turning point of the femoral metaphysis in all cases. CONCLUSIONS: In conclusion, the periosteum might support the hinge of MCWDFO within the area surrounded by the apex of the turning point of the femoral metaphysis and the upper border of the posterior part of the lateral femoral condyle.

Where and what damage occurs at the acromial undersurface in patients with rotator cuff tears?

BACKGROUND: The gross pathology of the acromial undersurface in shoulders with rotator cuff tears with subacromial impingement is not completely understood. Many researchers have focused on damage to the anterior one-third area of the acromial undersurface, but few have studied the middle and posterior one-third areas. The purpose of this study was to clarify where and what damage occurs at the acromial undersurface in patients with rotator cuff tears. METHODS: We performed arthroscopic shoulder (n = 182, all with rotator cuff tears; mean age, 64.9 ± 8.4 years) and cadaveric shoulder (n = 23, 14 intact cuffs and 9 rotator cuff tears; mean age, 74.8 years) evaluations to observe the extent and degree of damage to the acromial undersurface. We statistically analyzed the association between the severity of the damage to the acromial undersurface (assessed using the Copeland-Levy classification as A0, normal; A1, minor scuffing; A2, major damage; or A3, visualization of bare bone area) and rotator cuff tear size (assessed using the classification of DeOrio and Cofield as partial; small, <1 cm; medium, 1-3 cm; or large or massive, >3 cm). RESULTS: The anterior, middle, and posterior one-thirds of the acromial undersurface were somewhat damaged (class A1-A3) in 92.6%, 90.1%, and 78.6% of shoulders with rotator cuff tears, respectively, according to arthroscopic evaluation. Increasing cuff tear size was significantly associated with worsening degree of damage to the acromial undersurface (P < .001). In the 9 cadaveric shoulders with rotator cuff tears, class A1-A3 damage was identified in the anterior one-third area in 100%, in the middle one-third area in 88.9%, and in the posterior one-third area in 33.3%. In the 14 cadaveric shoulders with a normal rotator cuff, class A1-A3 damage was identified in the anterior one-third area in 35.7%, in the middle one-third area in 14.3%, and in the posterior one-third area in 0.71%. CONCLUSION: Damage to the acromial undersurface in patients with rotator cuff tears occurred at the middle, posterior, and anterior one-third areas, and the degree of damage was related to cuff tear size. Surgeons should evaluate the entire acromial undersurface to check for subacromial impingement damage at the middle and posterior one-third areas as well as the anterior one-third area of the acromial undersurface; this might aid in the treatment of patients with rotator cuff disease or subacromial impingement syndrome.

Effects of polyphenols on doxorubicin-induced oral keratinocyte cytotoxicity and anticancer potency against oral cancer cells.

BACKGROUND: Normal human oral keratinocytes are highly sensitive to anticancer drugs including doxorubicin. Resveratrol, epigallocatechin gallate, and tannic acid are polyphenolic compounds that were reported to have cardioprotective effect when combined with doxorubicin. However, it is unknown whether these polyphenols could protect normal human oral keratinocytes against doxorubicin-induced cytotoxicity without weakening its cytotoxic potential against oral cancer cells. Here, we examined the effects of the 3 polyphenolic compounds on doxorubicin-induced cytotoxicity in normal human oral keratinocytes and also investigated their effects on doxorubicin potency in HSC-2 human oral squamous cell carcinoma cells. METHODS: Cell viability was evaluated, followed by the analysis of apoptosis and necrosis. The changes in intracellular reactive oxygen species at the early stage after treatment were also examined. RESULTS: The results revealed that resveratrol in combination with doxorubicin additively augmented doxorubicin cytotoxicity in both types of cells. However, epigallocatechin gallate and tannic acid at a certain concentration mitigated the doxorubicin-induced keratinocyte toxicity mainly due to reduced doxorubicin-induced necrosis in normal human oral keratinocytes without weaken doxorubicin anticancer efficacy. The exact mechanism is still unknown but intracellular reactive oxygen species might be not the sole factor. CONCLUSIONS: This study for the first time reported the effects of resveratrol, epigallocatechin gallate, and tannic acid on doxorubicin-induced cytotoxicity in normal oral keratinocytes and oral cancer cells. The combined use of epigallocatechin gallate or tannic acid with doxorubicin at a certain concentration could mitigate doxorubicin-induced keratinocyte cytotoxicity without weakening doxorubicin anticancer efficacy.

Hyperthermia enhances bortezomib-induced apoptosis in human white blood cancer cells.

At present, the current therapeutic strategy for apoptosis induction mainly relies on the administration of pharmacological apoptotic modulators. Apart from that, apoptosis can be induced by various external stimuli such as hyperthermia, ionizing radiation, and electric fields. Despite advantages, both physical and pharmacological approaches bear some limitations as well. The rationale of this study was to overcome the limitations by combining hyperthermia and apoptotic modulator 'bortezomib' (Velcade). Two types of human blood cancer cell lines were utilized: human leukemic monocyte lymphoma cell U937 line and peripheral blood mononuclear cells (PMBCs) derived from the patient diagnosed with acute myeloid leukemia. Prior to apoptosis experiments, cytotoxicity tests were performed at three types of temperature regimes (40°, 42° and 44°C). We observed a gradual inhibition of cell viability correlating with an increase of temperature and drug concentration in both cell lines. However, there was no significant difference between sham group and groups of leukemic PMBCs treated by high temperature (44°C) and bortezomib. In U937 cells, combined treatment by heat shock and bortezomib led to an increase the number of cells underwent the late apoptosis stage. At the same time, similar treatment of PMBCs resulted in the stimulation of early apoptosis. Our data suggest that combination of bortezomib and hyperthermia enhances apoptosis induction in human cancer white blood cells, indicating a therapeutic potential for blood cancer therapy.

Induction of Apoptosis in U937 Cells by Using a Combination of Bortezomib and Low-Intensity Ultrasound.

BACKGROUND We scrutinized the feasibility of apoptosis induction in blood cancer cells by means of low-intensity ultrasound and the proteasome inhibitor bortezomib (Velcade). MATERIAL AND METHODS Human leukemic monocyte lymphoma U937 cells were subjected to ultrasound in the presence of bortezomib and the echo contrast agent Sonazoid. Two types of acoustic intensity (0.18 W/cm² and 0.05 W/cm²) were used for the experiments. Treated U937 cells were analyzed for viability and levels of early and late apoptosis. In addition, scanning electron microscopy analysis of treated cells was performed. RESULTS The percentage of cells that underwent early apoptosis in the group treated with ultrasound and Sonazoid was 8.0±1.31% (intensity 0.18 W/cm²) and 7.0±1.69% (0.05 W/cm²). However, coupling of bortezomib and Sonazoid resulted in an increase in the percentage of cells in the early apoptosis phase, up to 32.50±3.59% (intensity 0.18 W/cm²) and 33.0±4.90% (0.05 W/cm²). The percentage of U937 cells in the late apoptosis stage was not significantly different from that in the group treated with bortezomib only. CONCLUSIONS Our findings indicate the feasibility of apoptosis induction in blood cancer cells by using a combination of bortezomib, ultrasound contrast agents, and low-intensity ultrasound.

Soft Tissue Stabilization of the Hinge Position for Lateral Closing-Wedge Distal Femoral Osteotomy: An Anatomic Study.

Background: Soft tissue plays an important role in stabilizing the hinge point for osteotomy around the knee. However, insufficient data are available on the anatomic features of the soft tissue around the hinge position for lateral closing-wedge distal femoral osteotomy (LCWDFO). Purpose: To (1) anatomically analyze the soft tissue around the hinge position for LCWDFO, (2) histologically analyze the soft tissue based on the anatomic analysis results, and (3) radiologically define the appropriate hinge point to prevent unstable hinge fracture based on the results of the anatomic and histological analyses. Study Design: Descriptive laboratory study. Methods: In 20 cadaveric knees (age, 82.7 ± 7.8 years; range, 60-96 years), the soft tissue of the distal medial side of the femur was anatomically analyzed. The thicknesses of the periosteum and direct insertion of the adductor tendon (AT) were histologically examined and measured using an electron microscope. The thickness of the periosteum was visualized graphically, and the graph of the periosteum and radiograph of the knee were overlaid using image editing software. The appropriate hinge position was determined based on the periosteal thickness and attachment of the AT. Results: The mean thickness of the periosteum of the metaphysis was 352.7 ± 58.6 µm (range, 213.6-503.4 µm). The overlaid graph and radiograph revealed that the thickness of the periosteum changed at the part corresponding to the transition between the diaphyseal and metaphyseal ends of the femur. The mean width of the AT attached to the distal medial femur from the adductor tubercle toward the distal direction was 7.9 ± 1.3 mm (range, 6.3-9.7 mm). Conclusion: Results indicated that the periosteum and AT support the hinge for LCWDFO within the area surrounded by the apex of the adductor tubercle and the upper border of the posterior part of the lateral femoral condyle. Clinical Relevance: When the hinge point is located within the area surrounded by the apex of the adductor tubercle and the upper border of the posterior part of the lateral femoral condyle, these soft tissues work as stabilizers, and there is no risk of cutting into the joint space.

Intracranial Gene Delivery Mediated by Albumin-Based Nanobubbles and Low-Frequency Ultrasound.

Research in the field of high-intensity focused ultrasound (HIFU) for intracranial gene therapy has greatly progressed over the years. However, limitations of conventional HIFU still remain. That is, genes are required to cross the blood-brain barrier (BBB) in order to reach the neurological disordered lesion. In this study, we introduce a novel direct intracranial gene delivery method, bypassing the BBB using human serum albumin-based nanobubbles (NBs) injected through a less invasive intrathecal route via lumbar puncture, followed by intracranial irradiation with low-frequency ultrasound (LoFreqUS). Focusing on both plasmid DNA (pDNA) and messenger RNA (mRNA), our approach utilizes LoFreqUS for deeper tissue acoustic penetration and enhancing gene transfer efficiency. This drug delivery method could be dubbed as the "Spinal Back-Door Approach", an alternative to the "front door" BBB opening method. Experiments showed that NBs effectively responded to LoFreqUS, significantly improving gene transfer in vitro using U-87 MG cell lines. In vivo experiments in mice demonstrated significantly increased gene expression with pDNA; however, we were unable to obtain conclusive results using mRNA. This novel technique, combining albumin-based NBs and LoFreqUS offers a promising, efficient, targeted, and non-invasive solution for central nervous system gene therapy, potentially transforming the treatment landscape for neurological disorders.

[Molecular diagnosis and treatment using a new ultrasound contrast agent].

Ultrasound is often used for non-invasive diagnostic use in a clinical setting. Therapeutic ultrasound, on the other hand, has mainly been applied for its thermal or mechanical effects. Medical applications of high-energy ultrasound to ablate cancers are now under investigation(high intensity focused ultrasound: HIFU). Recently, there have been reports on the application of non-thermal ultrasound energy for treating various diseases in combination with drugs. Furthermore, the introduction of microbubbles, molecular targeted echo contrast agents, and carriers/enhancers of drugs has added a whole new dimension to diagnosis and therapy. Ultrasound irradiation of tissues and cells is effective for enhancing drug targeting, lowering systemic drug toxicity, and improving drug absorption rates. Efficient delivery of a drug into target cells or tissues for therapeutic purposes had always been a big challenge requiring multidisciplinary effort by experts in different scientific fields. Current advances in molecular imaging and therapy will also be discussed.

Efficient mRNA Delivery with Lyophilized Human Serum Albumin-Based Nanobubbles.

In this study, we developed an efficient mRNA delivery vehicle by optimizing a lyophilization method for preserving human serum albumin-based nanobubbles (HSA-NBs), bypassing the need for artificial stabilizers. The morphology of the lyophilized material was verified using scanning electron microscopy, and the concentration, size, and mass of regenerated HSA-NBs were verified using flow cytometry, nanoparticle tracking analysis, and resonance mass measurements, and compared to those before lyophilization. The study also evaluated the response of HSA-NBs to 1 MHz ultrasound irradiation and their ultrasound (US) contrast effect. The functionality of the regenerated HSA-NBs was confirmed by an increased expression of intracellularly transferred Gluc mRNA, with increasing intensity of US irradiation. The results indicated that HSA-NBs retained their structural and functional integrity markedly, post-lyophilization. These findings support the potential of lyophilized HSA-NBs, as efficient imaging, and drug delivery systems for various medical applications.

Use of ultrasound in drug delivery systems: emphasis on experimental methodology and mechanisms.

Recent studies have shown that ultrasound energy could be applied for targeting or controlling drug release. This new concept of therapeutic ultrasound combined with drugs has induced a great amount of interest in various medical fields. In this paper, several experimental systems are cited in which ultrasound is being utilized to evaluate new application of this modality. The mechanisms of ultrasound-mediated drug delivery are discussed in addition to the review of current advances in the use of ultrasound in systems involving research in cancer therapy, gene therapy, microbubbles and other drug delivery in vitro and in vivo experiments.

Ultrasound-Induced DNA Damage and Cellular Response: Historical Review, Mechanisms Analysis, and Therapeutic Implications.

The biological effects of ultrasound may be classified into thermal and nonthermal mechanisms. The nonthermal effects may be further classified into cavitational and noncavitational mechanisms. DNA damage induced by ultrasound is considered to be related to nonthermal cavitations. For this aspect, many in vitro studies on DNA have been conducted for evaluating the safety of diagnostic ultrasound, particularly in fetal imaging. Technological advancement in detecting DNA damage both in vitro and in vivo have elucidated the mechanism of DNA damage formation and their cellular response. Damage to DNA, and the residual damages after DNA repair are implicated in the biological effects. Here, we discuss the historical evidence of ultrasound on DNA damage and the mechanism of DNA damage formation both in vitro and in vivo, compared with those induced by ionizing radiation. We also offer a commentary on the safety of ultrasound over X-ray-based imaging. Also, understanding the various mechanisms involved in the bioeffects of ultrasound will lead us to alternative strategies for use of ultrasound for therapy.

Influence of Nanobubble Size Distribution on Ultrasound-Mediated Plasmid DNA and Messenger RNA Gene Delivery.

The use of nanobubbles (NBs) for ultrasound-mediated gene therapy has recently attracted much attention. However, few studies have evaluated the effect of different NB size distribution to the efficiency of gene delivery into cells. In this study, various size of albumin stabilized sub-micron bubbles were examined in an in vitro ultrasound (1 MHz) irradiation setup in the aim to compare and optimize gene transfer efficiency. Results with pDNA showed that gene transfer efficiency in the presence of NB size of 254.7 ± 3.8 nm was 2.5 fold greater than those with 187.3 ± 4.8 nm. Similarly, carrier-free mRNA transfer efficiency increased in the same conditions. It is suggested that NB size greater than 200 nm contributed more to the delivery of genes into the cytoplasm with ultrasound. Although further experiments are needed to understand the underlying mechanism for this phenomenon, the present results offer valuable information in optimizing of NB for future ultrasound-mediate gene therapy.

Metronomic irinotecan chemotherapy combined with ultrasound irradiation for a human uterine sarcoma xenograft.

Metronomic chemotherapy is the frequent administration of low doses of chemotherapeutic agents targeting tumor-associated endothelial cells. We examined the efficacy of metronomic irinotecan combined with low-intensity ultrasound (US) in human uterine sarcoma and evaluated its antiangiogenesis mechanism by measuring the circulating endothelial progenitor cells (CEP), a surrogate marker of angiogenesis. A human uterine sarcoma cell line, FU-MMT-3, was used in the present study because this tumor is one of the most malignant neoplasms of human solid tumors and it also has a high angiogenesis property. The combination of low-dose irinotecan and US irradiation significantly inhibited the tube formation of HUVEC and vascular endothelial growth factor expression of tumor cells in vitro. The FU-MMT-3 xenografts in nude mice were treated using US at a low intensity (2.0 w/cm(2), 1 MHz) for 4 min three times per week each after the intraperitoneal administration of irinotecan; this treatment was continued for 5 weeks. The tumor vascularity was assessed by contrast-enhanced color Doppler US in real time. The combination treatment significantly inhibited the mobilization of CEP and intratumoral vascularity compared with the control. This combination therapy showed a significant reduction in tumor volume, resulting in a significant prolongation of survival, in comparison with each treatment alone. These results suggest that the effect of metronomic chemotherapy for human uterine sarcoma was accelerated by US irradiation in vivo and this combination might therefore be potentially effective for new cancer therapy.

Optical observation of cell sonoporation with low intensity ultrasound.

Sonoporation is a promising drug delivery technique with great potential in medicine. However, its applications have been limited mostly by the lack of understanding its underlying biophysical mechanism, partly due to the inadequacy of the existing models for coupling with highly sensitive imaging techniques to directly observe the actual precursor events of cell-microbubble interaction under low intensity ultrasound. Here, we introduce a new in vitro method utilizing capillary-microgripping system and micro-transducer to achieve maximum level of experimental flexibility for capturing real time highly magnified images of cell-microbubble interaction, hitherto unseen in this context. Insonation of isolated single cells and microbubbles parallel with high speed microphotography and fluorescence microscopy allowed us to identify dynamic responses of cell-membrane/microbubble in correlation with sonoporation. Our results showed that bubble motion and linear oscillation in close contact with the cell membrane can cause local deformation and transient porosity in the cell membrane without rupturing it. This method can also be used as an in situ gene/drug delivery system of targeted cells for non-invasive clinical applications.

Ultrasound-mediated interferon ß gene transfection inhibits growth of malignant melanoma.

We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon ß (IFN-ß) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-ß in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-ß genes mixed with microbubbles. Successful sonotransfection with IFN-ß gene in vitro was confirmed by ELISA, which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-ß gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.

Differential cytotoxicity and sonosensitization by sanazole: effect of cell type and acoustic parameters.

PURPOSE: Although sanazole has been used as a hypoxic radiosensitizer, we recently reported on its ability to sensitize U937 cells to hyperthermia and X-irradiation under aerobic conditions, enhancing apoptotic cell death following the combined treatment. The current study was undertaken to evaluate the effect of sanazole as a sonosensitizer under previously studied acoustic conditions of different pulse repetition frequencies, using two cell lines representative of solid tumours and haematopoietic cancers. METHODS: Cells were treated with different doses of sanazole. Flow-cytometric analysis and DNA fragmentation assay were carried out at different times, and morphological features were also inspected. For ultrasound treatment, cells were pre-incubated with a non-cytotoxic dose of sanazole for 30 min before exposure. Evaluation of cell killing and a parallel examination of intracellular oxidative stress levels in both cell lines were performed using flow cytometry. RESULTS: Sanazole alone displayed selective cytotoxic effects towards solid tumour-derived cancer cells, resulting in complete cell death after 24 h of treatment, and enhanced the ultrasound-induced cell killing 6 h post-treatment. The enhancement seemed to be mediated by an additive increase in intracellular oxidative stress levels. CONCLUSION: Sanazole seems to be an efficient cytotoxic agent for the treatment of solid tumours and a promising sonosensitizer under aerobic conditions.

High-Intensity Focused Ultrasound in the Treatment of Breast Cancer.

High-intensity focused ultrasound (HIFU) is a non-invasive method of ablating malignant tumors. This paper will review the current clinical application of HIFU specially in the treatment of breast cancer. In addition to clinical studies, this review will also look into some basic studies that could address the technical issues related to this modality. In general, HIFU is considered to be safe being non-invasive and non-ionizing. The complication occurrence rate is low and repeated treatment is possible, making it an attractive option for some patients. However, for more than two decades since it was first used to treat breast cancer, clinical studies on HIFU still remain at the investigative stage and are only available in several centers. Reasons contributing to such few studies on HIFU include lack of specialized medical team and bioengineering technical staff, and breast cancer-dedicated imaging-HIFU platform to attain positive outcomes. Despite these circumstances, we believe that HIFU will eventually become the treatment of choice for most breast cancer patients in the near future.

Low-intensity ultrasound inhibits melanoma cell proliferation in vitro and tumor growth in vivo.

PURPOSE: To determine the effect of low-intensity ultrasound on cancer cell proliferation in vitro and tumor growth in vivo. METHODS: In vitro, several cancer cell lines were exposed to low-intensity ultrasound at 0.11 W/cm2 for 2 min. Of the cell lines screened, melanoma C32 is one of the cell lines that showed sensitivity to growth inhibition by ultrasound and was therefore used in succeeding experiments. In vivo, under the same ultrasound conditions used in vitro, C32 tumors in mice were exposed to ultrasound daily for 2 weeks, and the tumor volumes were monitored weekly using sonography. RESULTS: In vitro, C32 cell growth was inhibited, attaining 43.2% inhibition on the 3rd day. In vivo, tumor growth was significantly inhibited, with the treated tumors exhibiting 2.7-fold slowed tumor growth vs. untreated tumors at week 2. Such inhibition was not associated with increased cell death. Several genes related to the cell cycle and proliferation were among those significantly regulated. CONCLUSION: These findings highlight the potential of low-intensity ultrasound to inhibit tumor growth in a noninvasive, safe, and easy-to-administer way. In addition, this may suggest that the mechanical stress induced by ultrasound on C32 cells may have affected the intrinsic biomolecular mechanism related to the cell growth of this particular cell line. Further research is needed to identify which of the regulated genes played key roles in growth inhibition.

A study of the efficacy of ultrasonic waves in removing biofilms.

OBJECTIVE: The removal of adherent biofilms was assessed using ultrasonic waves in a non-contact mode. MATERIALS AND METHODS: In in vitro experiments, Streptococcus mutans (S. mutans) biofilms were exposed to ultrasonic waves at various frequencies (280 kHz, 1 MHz, or 2 MHz), duty ratios (0-90%), and exposure times (1-3 minutes), and the optimal conditions for biofilm removal were identified. Furthermore, the effect of adding a contrast medium, such as micro bubbles (Sonazoid), was examined. The spatial distribution and architecture of S. mutans biofilms before and after ultrasonic wave exposure were examined via scanning electron microscopy. The biofilm removal effect was also examined in in vivo experiments, using a custom-made oral cleaning device. RESULTS: When a 280 kHz probe was used, the biofilm-removing effect increased significantly compared to 1 and 2 MHz probes; more than 80% of the adherent biofilm was removed with a duty cycle of 50-90% and a 3 minutes exposure time. The maximum biofilm-removing effect was observed with a duty cycle of 80%. Furthermore, the addition of micro bubbles enhanced this biofilm-removing effect. In in vivo experiments, moderate biofilm removal was observed when a 280 kHz probe was used for 5 minutes. CONCLUSIONS: This study demonstrated that ultrasonic wave exposure in a non-contact mode effectively removed adherent biofilms composed of S. mutans in vitro.

Hypotonia-induced cell swelling enhances ultrasound-induced mechanical damage to cancer cells.

INTRODUCTION: It has been shown that killing of suspended cells by low-intensity ultrasound (0.08-0.11 W/cm(2)) can be enhanced by a mild non-lethal hypotonic (146 mOsm) medium. PURPOSE: In this study we wished to determine whether hypotonia-induced cell swelling of suspension cells was directly related to enhancement of ultrasound-mediated cell killing, and to verify whether similar effects could be observed on circulating and attached cells. METHODS: U937 cells under mild hypotonia were exposed to ultrasound for different times with real-time monitoring of cell size using a particle-size-distribution analyzer. To study the effect on attached cells, HeLa cells were exposed to ultrasound while under hypotonia in an in vivo-simulated set-up. RESULTS: The result showed that the enhanced cell killing (up to more than twice) was directly proportional to hypotonia-induced cell swelling. Similar membrane damage based on PI staining could be observed on HeLa cells treated with hypotonia. An in vivo-simulated circulating system also showed similar findings for hypotonia-enhanced ultrasound cell killing. CONCLUSION: These findings showed that mild hypotonia can be used to augment the effect of ultrasound in the treatment of cancers, particularly leukemia. The results showing that such enhancement is related to cell swelling could guide us toward proper timing of sonication while under hypotonic treatment.