Low-intensity therapeutic ultrasound: Role of frequency and its impact on the mechanosensitive signaling processes in chondrocytes

This dissertation primarily focuses on understanding the implications of ultrasonic frequency in low-intensity ultrasound (LIUS) mediated cellular processes. Our central hypothesis is that the ultrasonic frequency influences the key signaling events in the mechanotransduction pathway. Our hypothesis is predicated on the premise that for the optimal stimulation of cells via ultrasound (US), the frequency of an US signal must be maintained close to a natural resonant frequency of the cells. Our collective results provide evidence that the nuclear processes (i.e. transcription of load-inducible genes) responds to US with a frequency dependence, and it was maximized at 5.0 MHz. Our results also lend credence to the notion that LIUS promotes euchromatin (less condensed chromatin) formation to allow easier access of transcription factors to their target DNA sequences. Collectively, these findings suggest that the US signal is able to transverse a cell and directly impacts the nuclear machinery as a function of frequency. LIUS treated bovine articular chondrocytes (BACs) exhibited increased mRNA expression of SOX9 (master transcription factor for collagen-II). The LIUS mediated stimulation of BACs also induced the phosphorylation of Erk1/2 (MAP kinases with diverse cellular functions), upregulated the mRNA expression of hsp27 (small heat shock protein with cytoprotective function), activated the RhoA (a small GTPase known for regulating actin structure), and influenced the remodeling of actin microfilaments. Confocal images also depicted an increase in length and bending of the primary cilium (a “sensory cellular antenna” with mechanosensory function) in BACs upon US stimulation. These biochemical events are significant and represent potential signaling pathway(s) that can influence the regulation of SOX9 at the transcriptional level. To capitalize upon the positive bioeffects of US, an US assisted bioreactor (UBR) was fabricated. 2D-DIGE analysis of protein lysates from BACs subjected to US showed a differential expression of 138 unique proteins. In summary, this dissertation represents for the first time that the frequency content must be considered when investigating LIUS for therapeutic benefit and points towards potential signaling pathways of US mediated chondrogenesis. This may have far-reaching implications in optimizing the LIUS treatment conditions for cartilage tissue engineering