This study provides evidence that low-intensity ultrasound directly affects nuclear processes, and the magnitude of the effect varies with frequency. In particular, we show that the transcriptional induction of first load-inducible genes, which is independent of new protein synthesis, is frequency dependent. Bovine chondrocytes were exposed to low-intensity below the cavitational threshold) ultrasound at 2,5 and 8 MHz. Ultrasound elevated the expression of early response genes c-Fos, c-Jun and c-Myc, maximized at 5 MHz. The phosphorylated ERK inhibitor PD98059 abrogated any increase in c-series gene expression, suggesting that signaling occurs via the MAPPK/ERK pathway. However, phosphorylated ERK levels did not change with ultrasound frequency, indicating that processes downstream of ERK phosphorylation (such as nuclear transport and chromatin reorganization) respond to ultrasound with frequency dependence. A quantitative, biphasic mathematical model based on Biot theory predicted that cytoplasmic and nuclear stress is maximized at 5.2 ± 0.8 MHz for a chondrocyte, confirming experimental measurements.

No abstract

BackgroundContinuous low-intensity ultrasound (cLIUS) facilitates the chondrogenic differentiation of human mesenchymal stromal cells (MSCs) in the absence of exogenously added transforming growth factor-beta (TGFβ) by upregulating the expression of transcription factor SOX9, a master regulator of chondrogenesis. The present study evaluated the molecular events associated with the signaling pathways impacting SOX9 gene and protein expression under cLIUS.MethodsHuman bone marrow-derived MSCs were exposed to cLIUS stimulation at 14 kPa (5 MHz, 2.5 Vpp) for 5 min. The gene and protein expression of SOX9 was evaluated. The specificity of SOX9 upregulation under cLIUS was determined by treating the MSCs with small molecule inhibitors of select signaling molecules, followed by cLIUS treatment. Signaling events regulating SOX9 expression under cLIUS were analyzed by gene expression, immunofluorescence staining, and western blotting.ResultscLIUS upregulated the gene expression of SOX9 and enhanced the nuclear localization of SOX9 protein when compared to non-cLIUS-stimulated control. cLIUS was noted to enhance the phosphorylation of the signaling molecule ERK1/2. Inhibition of MEK/ERK1/2 by PD98059 resulted in the effective abrogation of cLIUS-induced SOX9 expression, indicating that cLIUS-induced SOX9 upregulation was dependent on the phosphorylation of ERK1/2. Inhibition of integrin and TRPV4, the upstream cell-surface effectors of ERK1/2, did not inhibit the phosphorylation of ERK1/2 and therefore did not abrogate cLIUS-induced SOX9 expression, thereby suggesting the involvement of other mechanoreceptors. Consequently, the effect of cLIUS on the actin cytoskeleton, a mechanosensitive receptor regulating SOX9, was evaluated. Diffused and disrupted actin fibers observed in MSCs under cLIUS closely resembled actin disruption by treatment with cytoskeletal drug Y27632, which is known to increase the gene expression of SOX9. The upregulation of SOX9 under cLIUS was, therefore, related to cLIUS-induced actin reorganization. SOX9 upregulation induced by actin reorganization was also found to be dependent on the phosphorylation of ERK1/2.ConclusionsCollectively, preconditioning of MSCs by cLIUS resulted in the nuclear localization of SOX9, phosphorylation of ERK1/2 and disruption of actin filaments, and the expression of SOX9 was dependent on the phosphorylation of ERK1/2 under cLIUS.Electronic supplementary materialThe online version of this article (10.1186/s13287-019-1532-2) contains supplementary material, which is available to authorized users.

Ultrasound at 5.0 MHz was noted to be chondro-inductive, with improved SOX-9 gene and COL2A1 protein expression in constructs that allowed for cell-to-cell contact. To achieve tissue-engineered cartilage using macroporous scaffolds, it is hypothesized that a combination of ultrasound at 5.0 MHz and transforming growth factor-β3 induces human mesenchymal stem cell differentiation to chondrocytes. Expression of miR-145 was used as a metric to qualitatively assess the efficacy of human mesenchymal stem cell conversion. Our results suggest that in group 1 (no transforming growth factor-β3, no ultrasound), as anticipated, human mesenchymal stem cells were not efficiently differentiated into chondrocytes, judging by the lack of decrease in the level of miR-145 expression. Human mesenchymal stem cells differentiated into chondrocytes in group 2 (transforming growth factor-β3, no ultrasound) and group 3 (transforming growth factor-β3, ultrasound) with group 3 having a 2-fold lower miR-145 when compared to group 2 at day 7, indicating a higher conversion to chondrocytes. Transforming growth factor-β3–induced chondrogenesis with and without ultrasound stimulation for 14 days in the ultrasound-assisted bioreactor was compared and followed by additional culture in the absence of growth factors. The combination of growth factor and ultrasound stimulation (group 3) resulted in enhanced COL2A1, SOX-9, and ACAN protein expression when compared to growth factor alone (group 2). No COL10A1 protein expression was noted. Enhanced cell proliferation and glycosaminoglycan deposition was noted with the combination of growth factor and ultrasound stimulation. These results suggest that ultrasound at 5.0 MHz could be used to induce chondrogenic differentiation of mesenchymal stem cells for cartilage tissue engineering.

No abstract

Human mesenchymal stem cells (hMSCs) hold great potential for cellular based therapeutics and tissue engineering applications and their expansion is an interesting prospect due to their low availability from in vivo sources. Therefore, this study investigated the effect of continuous-wave low-intensity ultrasound (LIUS) at 5.0-MHz and 14.0-kPa (<20 mW cm ) on the proliferative capacity, colony-formation efficiency, genetic stability, and differentiation potential of hMSCs. Additionally, potential signaling pathways involved in LIUS-mediated proliferation of hMSCs are studied. Compared to non-stimulated controls, LIUS-treated hMSCs shows a 1.9-fold greater colony-forming efficiency and 2.5-fold higher rate of cell proliferation, respectively. Differential staining and qRT-PCR analysis for selective chondrogenic, osteogenic, and adipogenic markers further confirmed that the LIUS treatment did not impact the multipotency of hMSCs. LIUS-treated hMSCs expressed normal male karyotype. The synthesis of cyclin-D1, a master regulator of cellular proliferation, is upregulated under LIUS and its enhanced mRNA expression under LIUS is noted to be mediated by the activation of both MAPK/ERK and PI3K/AKT pathways. In conclusion, LIUS promotes proliferation and self-renewal capacity of hMSCs.

A variety of mechanotransduction pathways mediate the response of fibroblasts or chondrocytes to ultrasound stimulation. In addition, regulatory pathways that co-ordinate stimulus-specific cellular responses are likely to exist. In this study, analysis was confined to the hypothesis that ultrasound stimulation (US) influences the chromatin structure, and that these changes may reflect a regulatory pathway that connects nuclear architecture, chromatin structure and gene expression. Murine fibroblasts seeded on tissue culture plates were stimulated with US (5.0 MHz (14 kPa), 51-s per application) and the thermal denaturation profiles of nuclei isolated from fibroblasts were assessed by dynamic scanning calorimetry (DSC). When compared to the thermal profiles obtained from the nuclei of non-stimulated cells, the nuclei obtained from stimulated cells showed a change in peak profiles and peak areas, which is indicative of chromatin remodeling. Independently, US was also observed to impact the histone (H1):chromatin association as measured indirectly by DAPI staining. Based on our work, it appears plausible that US can produce a remodeling of chromatin, thus triggering signal cascade and other intracellular mechanisms.

As cartilage does not have the innate potential to regenerate, translatable approaches based on mesenchymal stem cells (MSC) hold great promise for cartilage repair. In this study human MSC seeded in polyurethane scaffold were differentiated to chondrocytes in the presence or absence of dexamethasone (DEX), TGFβ3 and with or without exposure of ultrasound (US) at a resonant frequency of 5 MHz for the first two weeks according to the study groups: group 1 (DEX, No US), group 2 (DEX, US), group 3 (DEX, TGFβ3) and group 4 (DEX, TGFβ3, US). After two weeks, constructs were cultured in absence of any DEX and TGβ3 for another six weeks with or without US stimulation. At 8 weeks, group 2 constructs possessed dynamic stiffness (E * ), aggregate modulus (H a ) and permeability (k ) of 0.74±0.072 MPa, 0.14±0.003 MPa, 1.27×10 −10 ±3.501×10 −11 m 4 N −1 s −1 respectively with total glycosaminoglycan and collagen of 6.52±0.149 μg GAG μg −1 DNA and 17.14±0.642 μg COL μg −1 DNA, respectively. Synergism of US with TGFβ3 in group 4 led to a significant improvement (p<0.05) in the properties over group 2. Regression analysis showed that the combined biochemical properties correlated with the E * (R 2 ∼0.8-0.96). Ultrasound enhanced the protein expression of SOX9 and COL-II. Immunohistochemical and histological analyses revealed the presence of COL-II and GAG, while stained negative for von kossa and oil o red. In conclusion, this work has shown that: (a) MSC-seeding on polyurethane scaffolds, the subsequent lineage specific differentiation of MSC, and the further culture of MSC derived chondrocytes, were all performed in a seamless step in a 3D environment; notably under US stimulation in the absence of exogenous growth factors and (b) constructs under US had an aggregate modulus ranging from 0.14 to 0.17 MPa and approximating 1/10th the value of native cartilage.