MR-guided pulsed focused ultrasound improves mesenchymal stromal cell homing to the myocardium.

Image-guided pulsed focused ultrasound (pFUS) is a non-invasive technique that can increase tropism of intravenously (IV)-infused mesenchymal stromal cells (MSC) to sonicated tissues. MSC have shown promise for cardiac regenerative medicine strategies but can be hampered by inefficient homing to the myocardium. This study sonicated the left ventricles (LV) in rats with magnetic resonance imaging (MRI)-guided pFUS and examined both proteomic responses and subsequent MSC tropism to treated myocardium. T2-weighted MRI was used for pFUS targeting of the entire LV. pFUS increased numerous pro- and anti-inflammatory cytokines, chemokines, and trophic factors and cell adhesion molecules in the myocardial microenvironment for up to 48 hours post-sonication. Cardiac troponin I and N-terminal pro-B-type natriuretic peptide were elevated in the serum and myocardium. Immunohistochemistry revealed transient hypoxia and immune cell infiltration in pFUS-targeted regions. Myocardial tropism of IV-infused human MSC following pFUS increased twofold-threefold compared with controls. Proteomic and histological changes in myocardium following pFUS suggested a reversible inflammatory and hypoxic response leading to increased tropism of MSC. MR-guided pFUS could represent a non-invasive modality to improve MSC therapies for cardiac regenerative medicine approaches.

Ultrasound-Mediated Microbubble Destruction Suppresses Melanoma Tumor Growth.

Melanoma is one of the most aggressive types of cancer, and its incidence has increased rapidly in the past few decades. In this study, we investigated a novel treatment approach, the use of low-intensity ultrasound (2.3 W/cm2 at 1 MHz)-mediated Optison microbubble (MB) destruction (UMMD) to treat melanoma in a flank tumor model. The effect of UMMD was first evaluated in the melanoma cell line B16 F10 (B16) in vitro and then in mice inoculated with B16 cells. MB+B16 cells were exposed to US in vitro, resulting in significant cell death proportional to duty cycle (R2 = 0.74): approximately 30%, 50%, 80% and 80% cell death at 10%, 30%, 50% and 100% DC respectively. Direct implantation of tumors with MBs, followed by sonication, resulted in retarded tumor growth and improved survival (p = 0.0106). Immunohistochemical analyses confirmed the significant changes in expression of the cell proliferation marker Ki67 (p = 0.037) and a microtubule-associated protein 2 (p = 0.048) after US + MB treatment. These results suggest that UMMD could be used as a possible treatment approach in isolated melanoma and has the potential to translate to clinical trials.

Ultrasound-triggered PLGA microparticle destruction and degradation for controlled delivery of local cytotoxicity and drug release.

In this study, we investigated the low intensity ultrasound (US)-controlled delivery of local cytotoxicity and drug release via induced destruction and degradation of microparticles (MPs) made of poly(lactic-co-glycolic acid) (PLGA). This study was conducted in vitro with potential application towards tumor treatment in conjunction with direct injection. MPs, either loaded with or without doxorubicin (DOX), were prepared using a double-emulsion solvent-evaporation technique. First, the MPs were exposed to US with duty cycle (DC)-modulation. The destruction and degradation of MPs were evaluated using light and scanning electron microscopy. Second, the effects of US-mediated destruction/degradation of MPs on the local cytotoxicity as well as DOX release were evaluated. US-triggered MP destruction/degradation significantly enhanced nearby cell death and DOX release. These affects occurred in proportion to the DC. Our findings indicate that controlled cytotoxicity and DOX release by US could be useful in developing the minimally invasive therapeutic applications for tumor treatment.

Molecular and histological effects of MR-guided pulsed focused ultrasound to the rat heart.

BACKGROUND: Image-guided high intensity focused ultrasound has been used as an extracorporeal cardiac pacing tool and to enhance homing of stem cells to targeted tissues. However, molecular changes in the myocardium after sonication have not been widely investigated. Magnetic-resonance (MR)-guided pulsed focused ultrasound (pFUS) was targeted to the rat myocardium over a range of pressures and the microenvironmental and histological effects were evaluated over time. METHODS: Eight-to-ten-week-old Sprague-Dawley rats received T2-weighted MR images to target pFUS to the left ventricular and septum without cardiac or respiratory gating. Rats were sonicated through the thoracic wall at peak negative pressures (PNP) from 1 to 8 MPa at a center frequency of 1 MHz, 10 ms pulse duration and 1 Hz pulse repetition frequency for 100 pulses per focal target. Following pFUS, myocardium was harvested over 24 h and subjected to imaging, proteomic, and histological measurements. RESULTS: pFUS to the myocardium increased expression of cytokines, chemokines, and trophic factors characterized by an initial increase in tumor necrosis factor (TNF)-α followed by increases in pro- and anti-inflammatory factors that returned to baseline by 24 h. Immediately after pFUS, there was a transient (< 1 h) increase in N-terminal pro b-type natriuretic peptide (NT-proBNP) without elevation of other cardiac injury markers. A relationship between PNP and expression of TNF-α and NT-proBNP was observed with significant changes (p < 0.05 ANOVA) ≥ 4 MPa compared to untreated controls. Contrast-enhanced ex vivo T1-weighted MRI revealed vascular leakage in sonicated myocardium that was accompanied by the presence of albumin upon immunohistochemistry. Histology revealed infiltration of neutrophils and macrophages without morphological myofibril changes in sonicated tissue accompanied by pulmonary hemorrhage at PNP > 4 MPa. CONCLUSIONS: MR-guided pFUS to myocardium induced transient proteomic and histological changes. The temporal proteomic changes in the myocardium indicate a short-lived sterile inflammatory response consistent with ischemia or contusion. Further study of myocardial function and strain is needed to determine if pFUS could be developed as an experimental model of cardiac injury and chest trauma.

Characteristics of meniscus progenitor cells migrated from injured meniscus.

Serious meniscus injuries seldom heal and increase the risk for knee osteoarthritis; thus, there is a need to develop new reparative therapies. In that regard, stimulating tissue regeneration by autologous stem/progenitor cells has emerged as a promising new strategy. We showed previously that migratory chondrogenic progenitor cells (CPCs) were recruited to injured cartilage, where they showed a capability in situ tissue repair. Here, we tested the hypothesis that the meniscus contains a similar population of regenerative cells. Explant studies revealed that migrating cells were mainly confined to the red zone in normal menisci: However, these cells were capable of repopulating defects made in the white zone. In vivo, migrating cell numbers increased dramatically in damaged meniscus. Relative to non-migrating meniscus cells, migrating cells were more clonogenic, overexpressed progenitor cell markers, and included a larger side population. Gene expression profiling showed that the migrating population was more similar to CPCs than other meniscus cells. Finally, migrating cells equaled CPCs in chondrogenic potential, indicating a capacity for repair of the cartilaginous white zone of the meniscus. These findings demonstrate that, much as in articular cartilage, injuries to the meniscus mobilize an intrinsic progenitor cell population with strong reparative potential. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1966-1972, 2017.

Low-intensity pulsed ultrasound promotes chondrogenic progenitor cell migration via focal adhesion kinase pathway.

Low-intensity pulsed ultrasound (LIPUS) has been studied frequently for its beneficial effects on the repair of injured articular cartilage. We hypothesized that these effects are due to stimulation of chondrogenic progenitor cell (CPC) migration toward injured areas of cartilage through focal adhesion kinase (FAK) activation. CPC chemotaxis in bluntly injured osteochondral explants was examined by confocal microscopy, and migratory activity of cultured CPCs was measured in transwell and monolayer scratch assays. FAK activation by LIPUS was analyzed in cultured CPCs by Western blot. LIPUS effects were compared with the effects of two known chemotactic factors: N-formyl-methionyl-leucyl-phenylalanine (fMLF) and high-mobility group box 1 (HMGB1) protein. LIPUS significantly enhanced CPC migration on explants and in cell culture assays. Phosphorylation of FAK at the kinase domain (Tyr 576/577) was maximized by 5 min of exposure to LIPUS at a dose of 27.5 mW/cm(2) and frequency of 3.5 MHz. Treatment with fMLF, but not HMBG1, enhanced FAK activation to a degree similar to that of LIPUS, but neither fMLF nor HMGB1 enhanced the LIPUS effect. LIPUS-induced CPC migration was blocked by suppressing FAK phosphorylation with a Src family kinase inhibitor that blocks FAK phosphorylation. Our results imply that LIPUS might be used to promote cartilage healing by inducing the migration of CPCs to injured sites, which could delay or prevent the onset of post-traumatic osteoarthritis.

Inhibition of cell-matrix adhesions prevents cartilage chondrocyte death following impact injury.

Focal adhesions are transmembrane protein complexes that attach chondrocytes to the pericellular cartilage matrix and in turn, are linked to intracellular organelles via cytoskeleton. We previously found that excessive compression of articular cartilage leads to cytoskeleton-dependent chondrocyte death. Here we tested the hypothesis that this process also requires integrin activation and signaling via focal adhesion kinase (FAK) and Src family kinase (SFK). Osteochondral explants were treated with FAK and SFK inhibitors (FAKi, SFKi, respectively) for 2 h and then subjected to a death-inducing impact load. Chondrocyte viability was assessed by confocal microscopy immediately and at 24 h post-impact. With no treatment immediate post-impact viability was 59%. Treatment with 10 µM SFKi, 10 µM, or 100 µM FAKi improved viability to 80%, 77%, and 82%, respectively (p < 0.05). After 24 h viability declined to 34% in controls, 48% with 10 µM SFKi, 45% with 10 µM FAKi, and 56% with 100 µM FAKi (p < 0.01) treatment. These results confirmed that most of the acute chondrocyte mortality was FAK- and SFK-dependent, which implicates integrin-cytoskeleton interactions in the death signaling pathway. Together with previous findings, these data support the hypothesis that the excessive tissue strains accompanying impact loading induce death via a pathway initiated by strain on cell adhesion receptors.