Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.
Back

Tissue Engineering

Tissue Engineering - Poster Session A

Poster L3 - Cytomorphological Effect of Low-Frequency Low-Intensity Ultrasound on Angiogenesis of Endothelial Cells

Thursday, October 24, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  The goal of the work was to determine the effects of therapeutic ultrasound on endothelial morphological changes in 3D as an indicator of angiogenesis. Therapeutic application of low-frequency ( < 100kHz), low-intensity ( < 100mW/cm2) ultrasound (LFLI US) has been found to significantly enhance healing rate of chronic wounds (Samuels et al, 2013). Taking more than three months to heal on average, chronic wounds not only cause physical pain to patients but also heavy financial burdens (Iqbal et al, 2017). Hence, ultrasound treatment poses as a promising therapeutic to accelerate chronic wound healing; however, the exact biological mechanism behind this process is unknown (Samuels et al, 2013). We hypothesized that LFLI US promotes angiogenesis, the creation of new blood vessels, as the possible biological mechanism of ultrasound-assisted healing. Therefore, this study aims to investigate the cytomorphological effect of LFLI US on endothelial cells to observe any pro-angiogenic morphological indicators (Marcia et al, 2000).

Materials and

Methods:  500µL of Corning Matrigel basement membrane matrix (LDEV free) were thawed and cured per well on a 12-well plate (n=4). 600,000 HUVECs were seeded on each Matrigel with 2mL of vascular media. After 3 hours, the following ultrasound treatments were applied to each group for 15 minutes: control, 50mW/cm2, 100mW/cm2, and 150mW/cm2. Under LAS EZ brightfield microscope, 10 images of x5 magnification per group were taken at 0 hours, 10 hours, and 16 hours after ultrasound treatment. 5 representative images per group were then chosen and analyzed by ImageJ for the following measurements: number of junctions, number of meshes, length of tubule, and area of meshes. Tubule length was defined as the distance between two adjacent junctions connected by HUVEC network. A two-way ANOVA followed by a Tukey HSD test was used to determine significant differences. A p-value of less than 0.05 was considered significant.

Results, Conclusions, and Discussions: Ultrasound was found to have a significant effect on all quantified metrics: average mesh size, total tubule length, number of meshes, and number of junctions (p < 0.001), and on average tubule length (p < 0.05) (Figure 1). Time also had a significant impact on morphology in all metrics (p < 0.001) except for the number of meshes. Most of the significant differences were found between control or 50mW/cm2 and 100mW/cm2 or 150mW/cm2 groups at 10-hours and 16-hours following ultrasound exposure (Figure 1).
These data suggest that therapeutic ultrasound operating at 100mW/cm2 and 150mW/cm2 stimulates pro-angiogenic morphology more than in control and 50mW/cm2 intensities. 100mW/cm2 and 150mW/cm2 groups had significantly smaller mesh size and higher number of meshes than the control group at 16 hours, which indicates increased formation of vessels as new meshes tend to start small (Fig.1A). 100mW/cm2group also have significantly shorter average tubule lengths than 50mW/cm2 as HUVECs undergoing angiogenesis tend to have shorter tubules with frequent branching (Fig.1B) (Marcia et al, 2000). 100mW/cm2and 150mW/cm2 groups had significantly longer total tubule length than the control group (Fig.1C); there are generally greater number of meshes and junctions in 100mW/cm2 and 150mW/cm2 in comparison to control and 50mW/cm2 groups (Fig.1D,1E) - all of which are indications of pro-angiogenic behavior (Goodwin, 2007). However, it is noted that actual vessel formation was not a subject of observation in this experiment due to the short 24-hour time constraint of using Matrigel. It is possible that LFLI US may have increased the stiffness of extracellular matrix, stimulated by external mechanical force, which enhances cell adhesion and migration part of angiogenesis (Kretschmer et al, 2021). These data suggest that ultrasound may stimulate angiogenesis as a possible biological mechanism of ultrasound-assisted healing.
Based on the data acquired, this study strongly suggests that LFLI US of 100mW/cm2 and 150mW/cm2 intensity induced pro-angiogenic changes in HUVEC cytomorphology. Future work will be conducted with different cell density and possibly different assays such as fibrin gel bead and collagen gel instead of Matrigel.

Acknowledgements (Optional):