Focused Low-Intensity Pulsed Ultrasound (FLIPUS) Mitigates Apoptosis of MLO-Y4 Osteocyte-like Cells.

Long cytoplasmic processes of osteocytes orchestrate bone activity by integration of biochemical and mechanical signals and regulate load-induced bone adaptation. Low-Intensity Pulsed Ultrasound (LIPUS) is a clinically used technique for fracture healing that delivers mechanical impulses to the damaged bone tissue in a non-invasive and non-ionizing manner. The mechanism of action of LIPUS is still controversially discussed in the scientific community. In this study, the effect of focused LIPUS (FLIPUS) on the survival of starved MLO-Y4 osteocytes was investigated in vitro. Osteocytes stimulated for 10 min with FLIPUS exhibited extended dendrites, which formed frequent connections to neighboring cells and spanned longer distances. The sonicated cells displayed thick actin bundles and experienced increase in expression of connexin 43 (Cx43) proteins, especially on their dendrites, and E11 glycoprotein, which is responsible for the elongation of cellular cytoplasmic processes. After stimulation, expression of cell growth and survival genes as well as genes related to cell-cell communication was augmented. In addition, cell viability was improved after the sonication, and a decrease in ATP release in the medium was observed. In summary, FLIPUS mitigated apoptosis of starved osteocytes, which is likely related to the formation of the extensive dendritic network that ensured cell survival.

A Tissue Engineering Acoustophoretic (TEA) Set-up for the Enhanced Osteogenic Differentiation of Murine Mesenchymal Stromal Cells (mMSCs).

Quickly developing precision medicine and patient-oriented treatment strategies urgently require novel technological solutions. The randomly cell-populated scaffolds usually used for tissue engineering often fail to mimic the highly anisotropic characteristics of native tissue. In this work, an ultrasound standing-wave-based tissue engineering acoustophoretic (TEA) set-up was developed to organize murine mesenchymal stromal cells (mMSCs) in an in situ polymerizing 3-D fibrin hydrogel. The resultant constructs, consisting of 17 cell layers spaced at 300 µm, were obtained by continuous wave ultrasound applied at a 2.5 MHz frequency. The patterned mMSCs preserved the structured behavior within 10 days of culturing in osteogenic conditions. Cell viability was moderately increased 1 day after the patterning; it subdued and evened out, with the cells randomly encapsulated in hydrogels, within 21 days of culturing. Cells in the structured hydrogels exhibited enhanced expression of certain osteogenic markers, i.e., Runt-related transcription factor 2 (RUNX2), osterix (Osx) transcription factor, collagen-1 alpha1 (COL1A1), osteopontin (OPN), osteocalcin (OCN), and osteonectin (ON), as well as of certain cell-cycle-progression-associated genes, i.e., Cyclin D1, cysteine-rich angiogenic inducer 61 (CYR61), and anillin (ANLN), when cultured with osteogenic supplements and, for ANLN, also in the expansion media. Additionally, OPN expression was also augmented on day 5 in the patterned gels cultured without the osteoinductive media, suggesting the pro-osteogenic influence of the patterned cell organization. The TEA set-up proposes a novel method for non-invasively organizing cells in a 3-D environment, potentially enhancing the regenerative properties of the designed anisotropic constructs for bone healing.

Functional regulation of YAP mechanosensitive transcriptional coactivator by Focused Low-Intensity Pulsed Ultrasound (FLIPUS) enhances proliferation of murine mesenchymal precursors.

Yes-associated protein (YAP) acts as a mechanotransducer in determining the cell fate of murine C2C12 mesenchymal precursors as investigated after stimulation with ultrasound. We applied Focused Low-Intensity Pulsed Ultrasound (FLIPUS) at a sound frequency of 3.6 MHz, 100 Hz pulse repetition frequency (PRF), 27.8% duty cycle (DC), and 44.5 mW/cm2 acoustic intensity ISATA for 5 minutes and evaluated early cellular responses. FLIPUS decreased the level of phosphorylated YAP on Serine 127, leading to higher levels of active YAP in the nucleus. This in turn enhanced the expression of YAP-target genes associated with actin nucleation and stabilization, cytokinesis, and cell cycle progression. FLIPUS enhanced proliferation of C2C12 cells, whereas silencing of YAP expression abolished the beneficial effects of ultrasound. The expression of the transcription factor MyoD, defining cellular myogenic differentiation, was inhibited by mechanical stimulation. This study shows that ultrasound exposure regulates YAP functioning, which in turn improves the cell proliferative potential, critical for tissue regeneration process.

Influence of Donor Age and Stimulation Intensity on Osteogenic Differentiation of Rat Mesenchymal Stromal Cells in Response to Focused Low-Intensity Pulsed Ultrasound.

A focused low-intensity pulsed ultrasound (FLIPUS) was used to investigate the effects of stimulation period, acoustic intensity and donor age on the osteogenic differentiation potential of rat mesenchymal stromal cells (rMSCs). rMSCs from 3- and 12-mo-old female Sprague Drawly rats were isolated from bone marrow and stimulated 20 min/d with either 11.7 or 44.5 mW/cm2 (spatial average temporal average intensity) for 7 or 14 d. Osteogenic differentiation markers, i.e., Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and degree of matrix calcification were analyzed. On day 7 of stimulation, OCN gene expression was enhanced 1.9-fold in cells from young rats when stimulated with low intensity. The low intensity also led to a 40% decrease in RUNX2 expression on day 7 in aged cells, whereas high intensity enhanced expression of RUNX2 on day 14. FLIPUS treatment with low intensity resulted in a 15% increase in extracellular matrix mineralization in young but not old rMSCs. These differences suggest the necessity of a donor-age related optimization of stimulation parameters.

An investigation of BMP-7 mediated alterations to BMP signalling components in human tenocyte-like cells.

The incidence of tendon re-tears post-surgery is an ever present complication. It is suggested that the application of biological factors, such as bone morphogenetic protein 7 (BMP-7), can reduce complication rates by promoting tenogenic characteristics in in vitro studies. However, there remains a dearth of information in regards to the mechanisms of BMP-7 signalling in tenocytes. Using primary human tenocyte-like cells (hTLCs) from the supraspinatus tendon the BMP-7 signalling pathway was investigated: induction of the BMP associated Smad pathway and non-Smad pathways (AKT, p38, ERK1/2 and JNK); alterations in gene expression of BMP-7 associated receptors, Smad pathway components, Smad target gene (ID1) and tenogenic marker scleraxis. BMP-7 increases the expression of specific BMP associated receptors, BMPR-Ib and BMPR-II, and Smad8. Additionally, BMP-7 activates significantly Smad1/5/8 and slightly p38 pathways as indicated by an increase in phosphorylation and proven by inhibition experiments, where p-ERK1/2 and p-JNK pathways remain mainly unresponsive. Furthermore, BMP-7 increases the expression of the Smad target gene ID1, and the tendon specific transcription factor scleraxis. The study shows that tenocyte-like cells undergo primarily Smad8 and p38 signalling after BMP-7 stimulation. The up-regulation of tendon related marker genes and matrix proteins such as Smad8/9, scleraxis and collagen I might lead to positive effects of BMP-7 treatment for rotator cuff repair, without significant induction of osteogenic and chondrogenic markers.

Activation of Mechanosensitive Transcription Factors in Murine C2C12 Mesenchymal Precursors by Focused Low-Intensity Pulsed Ultrasound (FLIPUS).

In this paper, we investigated the mechanoresponse of C2C12 mesenchymal precursor cells to focused low-intensity pulsed ultrasound (FLIPUS). The setup has been developed for in vitro stimulation of adherent cells in the defocused far field of the ultrasound propagating through the bottom of the well plate. Twenty-four-well tissue culture plates, carrying the cell monolayers, were incubated in a temperature-controlled water tank. The ultrasound was applied at 3.6-MHz frequency, pulsed at 100-Hz repetition frequency with a 27.8% duty cycle, and calibrated at an output intensity of ISATA = 44.5 ±7.1 mW/cm2. Numerical sound propagation simulations showed no generation of standing waves in the well plate. The response of murine C2C12 cells to FLIPUS was evaluated by measuring activation of mechanosensitive transcription factors, i.e., activator protein-1 (AP-1), specificity protein 1 (Sp1), and transcriptional enhancer factor (TEAD), and expression of mechanosensitive genes, i.e., c-fos, c-jun, heparin binding growth associated molecule (HB-GAM), and Cyr-61. FLIPUS induced 50% ( p ≤ 0.05 ) and 70% ( p ≤ 0.05 ) increases in AP-1 and TEAD promoter activities, respectively, when stimulated for 5 min. The Sp1 activity was enhanced by about 20% ( p ≤ 0.05 ) after 5-min FLIPUS exposure and the trend persisted for 30-min ( p ≤ 0.05 ) and 1-h ( p ≤ 0.05 ) stimulation times. Expressions of mechanosensitive genes c-fos ( p ≤ 0.05 ), c-jun ( p ≤ 0.05 ), HB-GAM ( p ≤ 0.05 ), and cystein-rich protein 61 ( p ≤ 0.05 ) were enhanced in response to 5-min FLIPUS stimulation. The increase in proliferation of C2C12s occurred after the FLIPUS stimulation ( p ≤ 0.05 ), with AP-1, Sp1, and TEAD possibly regulating the observed cellular activities.

A Focused Low-Intensity Pulsed Ultrasound (FLIPUS) System for Cell Stimulation: Physical and Biological Proof of Principle.

Quantitative ultrasound (QUS) is a promising technique for bone tissue evaluation. Highly focused transducers used for QUS also have the capability to be applied for tissue-regenerative purposes and can provide spatially limited deposition of acoustic energy. We describe a focused low-intensity pulsed ultrasound (FLIPUS) system, which has been developed for the stimulation of cell monolayers in the defocused far field of the transducer through the bottom of the well plate. Tissue culture well plates, carrying the cells, were incubated in a special chamber, immersed in a temperature-controlled water tank. A stimulation frequency of 3.6 MHz provided an optimal sound transmission through the polystyrene well plate. The ultrasound was pulsed for 20 min daily at 100-Hz repetition frequency with 27.8% duty cycle. The calibrated output intensity corresponded to I(SATA) = 44.5 ± 7.1 mW/cm2, which is comparable to the most frequently reported nominal output levels in LIPUS studies. No temperature change by the ultrasound exposure was observed in the well plate. The system was used to stimulate rat mesenchymal stem cells (rMSCs). The applied intensity had no apoptotic effect and enhanced the expression of osteogenic markers, i.e., osteopontin (OPN), collagen 1 (Col-1), the osteoblast-specific transcription factor-Runx-2 and E11 protein, an early osteocyte marker, in stimulated cells on day 5. The proposed FLIPUS setup opens new perspectives for the evaluation of the mechanistic effects of LIPUS.

Stimulation of Bone Repair with Ultrasound.

This chapter reviews the different options available for the use of ultrasound in the enhancement of fracture healing or in the reactivation of a failed healing process: LIPUS, shock waves and ultrasound-mediated delivery of bioactive molecules, such as growth factors or plasmids. The main emphasis is on LIPUS, or Low Intensity Pulsed Ultrasound, the most widespread and studied technique. LIPUS has pronounced bioeffects on tissue regeneration, while employing intensities within a diagnostic range. The biological response to LIPUS is complex as the response of numerous cell types to this stimulus involves several pathways. Known to-date mechanotransduction pathways involved in cell responses include MAPK and other kinases signaling pathways, gap-junctional intercellular communication, up-regulation and clustering of integrins, involvement of the COX-2/PGE2 and iNOS/NO pathways, and activation of the ATI mechanoreceptor. Mechanisms at the origin of LIPUS biological effects remain intriguing, and analysis is hampered by the diversity of experimental systems used in-vitro. Data point to clear evidence that bioeffects can be modulated by direct and indirect mechanical effects, like acoustic radiation force, acoustic streaming, propagation of surface waves, heat, fluid-flow induced circulation and redistribution of nutrients, oxygen and signaling molecules. One of the future engineering challenge is therefore the design of dedicated experimental set-ups allowing control of these different mechanical phenomena, and to relate them to biological responses. Then, the derivation of an 'acoustic dose' and the cross-calibration of the different experimental systems will be possible. Despite this imperfect knowledge of LIPUS biophysics, the clinical evidence, although most often of low quality, speaks in favor of the clinical use of LIPUS, when the economics of nonunion and the absence of toxicity of this ultrasound technology are taken into account.

Stimulation of bone repair with ultrasound: a review of the possible mechanic effects.

In vivo and in vitro studies have demonstrated the positive role that ultrasound can play in the enhancement of fracture healing or in the reactivation of a failed healing process. We review the several options available for the use of ultrasound in this context, either to induce a direct physical effect (LIPUS, shock waves), to deliver bioactive molecules such as growth factors, or to transfect cells with osteogenic plasmids; with a main focus on LIPUS (or Low Intensity Pulsed Ultrasound) as it is the most widespread and studied technique. The biological response to LIPUS is complex as numerous cell types respond to this stimulus involving several pathways. Known to-date mechanotransduction pathways involved in cell responses include MAPK and other kinases signaling pathways, gap-junctional intercellular communication, up-regulation and clustering of integrins, involvement of the COX-2/PGE2, iNOS/NO pathways and activation of ATI mechanoreceptor. The mechanisms by which ultrasound can trigger these effects remain intriguing. Possible mechanisms include direct and indirect mechanical effects like acoustic radiation force, acoustic streaming, and propagation of surface waves, fluid-flow induced circulation and redistribution of nutrients, oxygen and signaling molecules. Effects caused by the transformation of acoustic wave energy into heat can usually be neglected, but heating of the transducer may have a potential impact on the stimulation in some in-vitro systems, depending on the coupling conditions. Cavitation cannot occur at the pressure levels delivered by LIPUS. In-vitro studies, although not appropriate to identify the overall biological effects, are of great interest to study specific mechanisms of action. The diversity of current experimental set-ups however renders this analysis very complex, as phenomena such as transducer heating, inhomogeneities of the sound intensity in the near field, resonances in the transmission and reflection through the culture dish walls and the formation of standing waves will greatly affect the local type and amplitude of the stimulus exerted on the cells. A future engineering challenge is therefore the design of dedicated experimental set-ups, in which the different mechanical phenomena induced by ultrasound can be controlled. This is a prerequisite to evaluate the biological effects of the different phenomena with respect to particular parameters, like intensity, frequency, or duty cycle. By relating the variations of these parameters to the induced physical effects and to the biological responses, it will become possible to derive an 'acoustic dose' and propose a quantification and cross-calibration of the different experimental systems. Improvements in bone healing management will probably also come from a combination of ultrasound with a 'biologic' components, e.g. growth factors, scaffolds, gene therapies, or drug delivery vehicles, the effects of which being potentiated by the ultrasound.