The Effect of Low-Intensity Pulsed Ultrasound on Extracellular Matrix and its cross-linking and organisation.

Abstract

Chronic wounds are characterised by their inability to heal within the expected time frame, significantly impacting patient welfare and even life expectancy, while also imposing considerable financial burdens on the healthcare system. The extracellular matrix (ECM) plays an essential role by interacting with growth factors and providing the physical substrate over which cells migrate during healing. However, in chronic wounds, the ECM components often appear to be dysfunctional. Low-intensity ultrasound, as a form of mechanical stimulation, has shown promising results at the cellular level. This project aims to investigate the effect of Low-Intensity Pulsed Ultrasound (LIPUS) on gene expression, matrix deposition, cross-linking, and the organisation of specific ECM molecules. In this study, two cell lines were used, human foreskin fibroblast (HFF) and telomerase-immortalized fibroblasts (TIF). Then, I performed western blot, Quantitative PCR, Immunofluorescence, migration assay and traction force microscopy to investigate the protein and gene expression changes, matrix alignment and contractility and cell migration after the LIPUS treatment. 1.5-MHz frequency, a 20% duty cycle pulse to generate a 30 mW/cm2 spatial average and temporal average (SATA) were applied to the treatment group. The treatment was performed for 20 minutes per day for five days. The results indicate that matrix composition was altered by LIPUS, with specific effects on ECM molecules involved in healing. Transforming growth factor-beta (TGF-β) and collagen type I showed a reduction in protein expression in the conditioned medium, whereas lysyl oxidase (LOX), collagen type III, and fibronectin exhibited increased expression. These findings reveal that LIPUS treatment changes the composition of secreted matrix by altering the expression profiles of several components and regulatory factors in a manner that is far more nuanced than simply promoting expression. LIPUS treatment also altered fibronectin fibre curvature, leading to more random alignment. This is significant as it suggests that a 20-minute treatment can have long-lasting healing effects by modifying ECM architecture. Traction Force Microscopy revealed reduced cellular contractility after LIPUS treatment, likely due to changes in TGF-β expression, which explains the altered ECM alignment. Overall, these findings show that LIPUS influences both ECM structure and cellular mechanical properties, potentially improving tissue repair and healing outcomes. Together, these findings indicate LIPUS's potential as a therapeutic tool to enhance wound healing and reduce the scar effect, particularly in cases of chronic wounds where ECM dysfunction is common. Also, this project suggests that LIPUS can affect ECM dynamics, showing changes in protein expression and ECM organisation.