Regina Puts

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 ISATA = 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.

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.

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.

Mechanosensitive response of murine C2C12 myoblasts to focused Low-Intensity Pulsed Ultrasound (FLIPUS) stimulation

Quantitative Ultrasound (QUS) is a promising technique for bone tissue evaluation. Highly focused transducers, used for QUS, have also a capability to be applied for tissue-regenerative purposes and can provide geometrically controlled deposition of an accurate acoustic dose. This allows for overcoming of unwanted artifacts, i.e. standing waves, ring interferences, temperature elevations, cross-stimulation of adjacent wells, which often lead to irreproducible biological results, associated with existing Low-Intensity Pulsed Ultrasound (LIPUS) in-vitro set-ups. Focused LIPUS (FLIPUS) is a novel reliable tool, which is minimizes or avoids the aforementioned artifacts. In this study we evaluated mechanical response of murine C2C12 myoblastic cells, possessing osteogenic-lineage differentiation potential, to the well-controlled FLIPUS dose. The optimized acoustic dose (3.6 MHz, pulsed at 100 Hz with 28% duty cycle, corresponding to ISATA = 29.4 ± 4.8 mW/cm2) induced 1.5 fold increase (p<;0.05) in AP-1 promoter activity already after 5 minutes of FLIPUS exposure. The Sp1 binding was enhanced by about 20 % (p<;0.05) after 5 min FLIPUS stimulation and the trend persisted for 30 min (p<;0.01) and 1 hour (p<;0.01) stimulation times. Activation of TEAD-binding sequences to about 2 fold (p<;0.01) was observed after 5 min FLIPUS stimulation. The proliferation of the C2C12 cells was up-regulated in starving conditions on day 1 and 3 of daily short-term FLIPUS-treatments. These results imply the pro-proliferative nature of the selected FLIPUS-dose in C2C12s, with potential functional role of the described mechanosensitive transcription factors. The use of focused ultrasound for both visualization and therapeutic purposes may be applied in clinical settings to provide monitoring and support healing of damaged tissues.

Activation of mechanosensitive transcription factors in murine C2C12 myoblasts by focused low-intensity pulsed Ultrasound (FLIPUS)

Quantitative Ultrasound (QUS) is a promising technique for bone tissue evaluation. Focused transducers used for QUS also have a capability to be applied for tissue-regenerative purposes and can provide spatially limited deposition of acoustic energy. We describe a focused LIPUS (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. The ultrasound was introduced at 3.6 MHz, pulsed at 100 Hz repetition frequency with 27.8% duty cycle. The calibrated output intensity corresponded to ISATA = 44.5 ± 7.1 mW/cm2. In this study we evaluated mechanical response of murine C2C12 myoblastic cells to ultrasound. FLIPUS induced 1.5 fold increase (p<;0.05) in AP-1 promoter activity already after 5-minute of FLIPUS exposure. The Sp1 binding was enhanced by about 20% (p<;0.05) after 5-min FLIPUS stimulation and the trend persisted for 30 min (p<;0.01) and 1 hour (p<;0.01) stimulation times. Increase of 70% in activation of TEAD-binding sequences (p<;0.01) was observed after the 5 min FLIPUS treatment. The proliferation of the C2C12 cells was up-regulated after the 5-minlong FLIPUS exposure. These results imply the pro-proliferative nature of the selected FLIPUS-intensity in C2C12s, with potential functional role of the described mechanosensitive transcription factors. The use of FLIPUS as a visualization and regenerative technique can be further applied in clinical settings as a device providing monitoring and healing of the damaged tissue.

In-vitro stimulation of cells of the musculoskeletal system with focused Low-Intensity Pulsed Ultrasound (FLIPUS): Analyses of cellular activities in response to the optimized acoustic dose

Low-Intensity Pulsed Ultrasound (LIPUS) is a safe treatment for fresh fractures, delayed- and non-union bone. Certain inconsistency is associated with the reported in-vitro data, which could be attributed to difficulties in reproducing similar conditions using unfocused transducers in experimental in-vitro set-ups. In our study we address the above issues by using focused LIPUS (FLIPUS), which avoids the previously described potential sources of physical side-effects and therefore, allows more robust “acoustic dose” definition and an “acoustic dose - observed effect” correlation in an in-vitro setting. In our experiments all relevant sound field parameters were measured and adjusted such that they provide an optimal and even sound transmission. Five days after daily FLIPUS stimulation, gene expression of osteopontin (OPN), Runx2, collagen-1 (Col-1), E11 antigen and dentin matrix protein-1 (DMP-1) was up-regulated in rat mesenchymal stem cells (rMSCs). These cells under reduced nutrient supply also showed higher proliferation rate on days 3 and 4. Enhanced proliferation on days 2, 3 and 4 was observed in murine MC3T3-E1 pre-osteoblasts after the FLIPUS treatment under similar conditions. Increased expressions of early response genes c-jun and cyclooxygenase-2 (COX-2) were observed 30 min post-FLIPUS-exposure in the same cells. FLIPUS induced enhanced phosphorylation of Smad1/5/8 (mothers against decapentaplegic) transcription factors in MC3T3-E1s, when Bone Morphogenetic Protein-2 (BMP-2) and FLIPUS were applied simultaneously. These results indicate that FLIPUS is a convenient set-up to study the mechanism of bone regeneration in response to ultrasound in vitro.

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.

Ultrasound for bone tissue engineering and regenerative medicine

We will review the role of ultrasound in bone tissue engineering and regenerative medicine, for enhancement of fracture healing and reactivation of a failed healing process. These approaches rely typically either on the direct stimulation of the healing process or in the delivery at the injury site of scaffold-embedded biologics such as cells, signaling molecules, and genetic material. Different options are available for ultrasound enhancement of fracture healing such as low intensity pulsed ultrasound (LIPUS) and shock waves, for the delivery of regenerative factors and encoding genes, and for the assembly and patterning of scaffolds. A main emphasis will be put on LIPUS-related bioeffects, the most widespread and studied technique. Pronounced bioeffects on tissue regeneration have been reported employing intensities within a diagnostic range. We will describe the current knowledge of the biological response to LIPUS, involving numerous cell types and molecular pathways, hypothesis about LIPUS biophysics...

51 Insights Into Bmp-7 Signalling Of Rotator Cuff Tenocytes

Introduction We reported previously that important cellular characteristics such as the cell activity and the expression and synthesis of collagen I can be strongly enhanced by stimulation of tenocytes with BMP-7 [Klatte-Schulz et al. 2014]. Since tendon healing after surgical reconstructions still represents a challenging topic, due to complications such as retears or non-healing of the tendon tissue, the augmentation of the healing using growth factors like BMP-7 may be a possible treatment option. What remains unknown is how BMP-7 signals in tenocytes, which was analysed in the present study. Methods Tenocytes of 6 male donors (63–69 years) isolated from biopsies of supraspinatus tendon tears were seeded in cell culture dishes, cultured for 3–5 days and stimulated with 200 or 1000 ng/ml rhBMP-7. Protein lysate samples were collected after 15, 30, 60 and 120 min and the phosphorylation of Smad1/5/8 was analysed using Western Blotting technique. RNA was isolated after 1, 2, 4 and 8 h from the cells. The gene expression of BMP-Receptors (BMPR-Ia, BMPR-Ib, BMPRII), Activin receptors (ActR-I, ActR-IIa, ActR-IIb), Smad 1, 4, 5, 8 and the BMP target gene ID1, as well as scleraxis, osteocalcin and collagen II was analysed by Real-Time PCR. Statistics: Mann-Whitney-U Test, p ≤ 0.05, Bonferroni Holm Correction Results The relative phosphorylation level of Smad1/5/8 was significantly increased 30 and 60 min after stimulation with BMP-7 compared to unstimulated control cells (Figure 1). Abstract 51 Figure 1 Smad phosphorylation relative to total Smad and GAPDH and normalized to unstimulated control The expression of BMPR-Ib was increased the most compared to both other type I receptors 4 and 8 h after stimulation. In the type II receptor group, BMPR-II showed the highest increase after 2 to 8 h, while ActR-IIb was down-regulated after 2 h and 4 h. The expression of Smad1 and 4 was slightly increased and Smad5 showed no increase of expression. Smad8 expression was strongly up-regulated 4 h and 8 h after stimulation (Figure 2). The expression of ID1 was increased already at 1 h and 2 h and strongly enhanced 8 h after stimulation. Scleraxis expression showed a strong increase at 1, 2 and 4 h and a massive increase at 8 h after stimulation. No change in the osteocalcin and collagen II expression was found after BMP-7 stimulation. Abstract 51 Figure 2 Smad8 expression relative to 18 s and normalised to control. Discussion The study showed that the type I receptor BMPR-Ib and the type II receptor BMPR-II seem to be the most regulated receptors on their mRNA level after tenocyte stimulation with BMP-7. Additionally, the receptor-regulated Smad8 seems to represent the essential signalling molecule in tenocytes after BMP-7 stimulation. An important role of Smad8 in tenogenic differentiation was reported previously. [Hoffmann et al., 2006] The strong increase of the tendon related gene scleraxis in the cells may underline the positive effect of BMP-7 treatment for rotator cuff repair. References Klatte-Schulz, et al. J Orthop Res. 2014;32(1):129–37 Hoffmann, et al. J Clin Invest. 2006;116(4):940–52