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High School Student with a Traditionally Underrepresented Background

HS Poster

Poster 105 - Low-frequency (20 kHz), Low-intensity (100 mW/cm2) Ultrasound Significantly Increases VEGF Secretion on Endothelial Cells Seeded in 3D Collagen Scaffold

Saturday, October 26, 2024
10:00 AM – 12:00 PM EST
Location: Exhibit Hall E, F & G

    Presenting Author(s)

  • MS

    Maya Sak

    Student Researcher
    Bucknell University, United States

    • Last Author(s)

  • Tobias Kim

    Undergraduate Researcher
    Bucknell University
    Lewisburg, Pennsylvania, United States

    • Co-Author(s)

  • Angelina L. Santos (she/her/hers)

    Undergraduate Researcher
    Bucknell University
    Lewisburg, Pennsylvania, United States

    • Primary Investigator(s)

  • Olivia Boerman, PhD (she/her/hers)

    Assistant Professor of Biomedical Engineering
    Bucknell University
    Lewisburg, Pennsylvania, United States


Introduction: The purpose of this research is to determine the effects of therapeutic ultrasound on angiogenesis via protein secretion analysis. In a pilot human study, it was found that therapeutic ultrasound significantly advanced chronic wound healing [1]. Samuels et al. showed venous ulcers treated with low frequency (20 - 100 kHz), low-intensity (100 mW/cm2) ultrasound for 15 minutes once a week led to significantly reduced wound area when compared to sham-treated subjects [1]. However, the biological mechanisms involved in therapeutic ultrasound-assisted wound healing are not understood. One potential mechanism may be through the promotion of angiogenesis. Angiogenesis is the formation of new blood vessels, and is shown to be a vital step in wound healing. The secretion of vascular endothelial growth factor (VEGF) has been shown to be a critical protein that stimulates angiogenesis [2]. Given the role of VEGF in wound healing, this research aims to analyze VEGF secretion of endothelial cells in a 3D collagen matrix following therapeutic ultrasound exposure.

Materials and

Methods: 600,000 human umbilical vein endothelial cells (HUVECs) were seeded in 3D collagen matrices, incubated for 48 hours and exposed to low-frequency, low-intensity ultrasound (LFLI US), at a frequency of 20 kHz for 15 minutes. Four experimental groups were established: HUVEC cells exposed to 50mW/cm2 LFLI US, 100mW/cm2, 150mW/cm2, and a sham group. Conditioned media was collected 24 hours following ultrasound exposure (n = 4). The sandwich ELISA protocol was used to evaluate the concentration of VEGF secreted by each experimental group. Each experimental group (sham, 50 mW/cm2, 100 mW/cm2, and 150 mW/cm2) was diluted in deionized water at a factor of 0.5. Following the manufacturer's protocol, data were collected at 405 nm and 650 nm and analyzed using MyAssays via Microsoft Excel. A one-way ANOVA was employed with Tukey’s post hoc analysis and a p-value of less than 0.05 was considered to be significant.

Results, Conclusions, and Discussions: Results following the ELISA protocol and one-way ANOVA indicate significance between the experimental groups (p < 0.001). In particular, results from Tukey's post-hoc analysis revealed that cells treated with 100mW/cm2 are significantly different from all other experimental groups, while the 50mW/cm2 and 150mW/cm2 groups show no significance relative to the sham group. This result is consistent with the current body of research, as Hu et al. found that therapeutic ultrasound (50 mW/cm2) promoted angiogenesis [3]. Also, given that the 100mW/cm2 group increased VEGF secretion more than the sham group, a positive correlation may be made between LFLI US and VEGF secretion, which suggests that ultrasound may advance the process of angiogenesis.

This research aimed to assess the role of VEGF secretion in wound healing via therapeutic ultrasound, using a 3D collagen model to more closely mimic physiological conditions. Findings from a sandwich ELISA suggest a positive correlation between LFLI US and angiogenesis via the secretion of VEGF. Furthermore, this research confirms previous findings showing that 100mW/cm2 is an optimal intensity with which to apply therapeutic ultrasound. Although these results are consistent with past findings, there are limitations to the experimental design. In future in vitro studies, HUVEC cells should be co cultured with other cell types to more accurately simulate in vivo conditions and another experimental group should be introduced to better test the null hypothesis.

Acknowledgements (Optional): This work was supported by the National Science Foundation (Award # 2347558).

References (Optional): [1] Samuels, J. A., Weingarten, M. S., Margolis, D. J., Zubkov, L., Sunny, Y., Bawiec, C. R., Conover, D., & Lewin, P. A. (2013). Low-frequency ( < 100 kHz), low-intensity ( < 100 mW/cm(2)) ultrasound to treat venous ulcers: a human study and in vitro experiments. The Journal of the Acoustical Society of America, 134(2), 1541–1547. https://doi.org/10.1121/1.4812875
[2] Cañedo-Dorantes, Luis, Cañedo-Ayala, Mara, Skin Acute Wound Healing: A Comprehensive Review, International Journal of Inflammation, 2019, 3706315, 15 pages, 2019. https://doi.org/10.1155/2019/3706315
[3] Hu, R., Yang, Z. Y., Li, Y. H., & Zhou, Z. (2022). LIPUS Promotes Endothelial Differentiation and Angiogenesis of Periodontal Ligament Stem Cells by Activating Piezo1. International journal of stem cells, 15(4), 372–383. https://doi.org/10.15283/ijsc22024