Z8 - Effects of Low-Frequency, Low-Intensity Ultrasound on the Secretion of IL-6 in 3D Macrophage-Endothelial Co-Culture

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Jacob T. Mudge

Undergraduate Research Assistant
Bucknell University
Kennett Square, Pennsylvania, United States

Primary Investigator(s)

Olivia Boerman

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

Co-Author(s)

Isaac S. Horst

Undergraduate Research Assistant
Bucknell University, Pennsylvania, United States
Max Clark

Undergraduate Research Assistant
Bucknell University
Lewisburg, Pennsylvania, United States

Introduction: : Chronic wounds remain stuck in the inflammatory stage of wound healing which results in disruption of the regenerative process and makes successful healing in chronic wounds difficult to achieve in many patients. The difficulty of chronic wound treatment results in extremely high costs: chronic wounds account for 25 billion dollars of spending in the United States alone [1]. Moreover, chronic wounds can also lead to serious complications such as amputations and an overall poorer quality of life for many patients.
Low-frequency, Low-intensity ultrasound (LFLI US) has been shown to significantly decrease healing time of chronic wounds when compared to sham-treated patients in clinical trials [1, 2]. Ultrasound treatment is portable, cheap, and effective all of which make it a promising therapy for chronic wounds. However, the underlying biological mechanisms behind ultrasound treatment in chronic wounds remain unknown.
Macrophages are immune cells that play an important role in regulating the stages of healing in acute wounds. Macrophages are primarily categorized by two different phenotypes: the M1 pro-inflammatory expression, and the M2 pro-healing expression [3]. M1 macrophages are most prevalent in the initial inflammatory phase of wound healing where they primarily phagocytose damaged cells and potentially harmful pathogens in order to clear the surrounding tissue for the proliferative and remodeling phases [3,4]. These actions are driven by inflammatory cytokines which upregulate the immune response and recruit additional immune cells [4]. Therefore, the goal of this work is to understand how ultrasound affects the secretion of IL-6 in a macrophage-endothelial co-culture.

Materials and

Methods: : Macrophage and Endothelial cell cultures were split and fed new media until they reached a quantity of at least 16 million and 9.6 million each. 600,000 Human Endothelial cells were seeded onto a 3D Surgifoam Collagen Matrix cut into 25 µm slices, approximately 96 hours later 1,000,00 macrophages were seeded into the collagen matrix. In both cases 50uL was pipetted onto both sides of the scaffold.
LFLI US was applied at intensities of 50mW/cm2 , 100mW/cm2 , and 150mW/cm2. Each well plate had a total of four tissue scaffolds inside, and ultrasound was applied using applicators wrapped in tegaderm. Well plates were coupled with water to account for ultrasound reflections and standing waves. Ultrasound was applied for an interval of 15 minutes each.
48 hours following ultrasound exposure, media from each well was collected and frozen. Thermofisher Il-6 sandwich ELISA was used in order to quantify the concentration of IL-6 protein in conditioned media. The ELISA plate was read at standard and correctional wavelengths of 450nm and 570nm respectively. Unknown concentrations were calculated using the standard curve, and a one way ANOVA Tukey’s post hoc analysis was performed on Graph Pad Prism. A p-value of less than 0.05 was considered to be significant.

Results, Conclusions, and Discussions:: We saw a significant decrease in IL-6 secretion in the 50 mW/cm2 group compared to the control (p < 0.01). Interestingly, the 100 and 150 mW/cm2 groups were not significantly different from the control group, but were significantly different from the 50 mW/cm2 (p < 0.01). . The data from the 50 mW/cm2 group supports our hypothesis that LFLI US may decrease pro-inflammatory cytokine secretion as a mechanism of ultrasound-mediated healing. Our data corroborates the previously published work by Qin et al. who show that ultrasound can stimulate M1 to M2 macrophage transition [5].
However, neither the 100mW/cm2 nor the 150mW/cm2 groups showed statistical significance. This data suggests unknown biological mechanisms that affect IL-6 at these ultrasound intensities and should be more rigorously tested in future investigations. Overall, our data demonstrates the novel finding that ultrasound significantly decreased IL-6 protein secretion in a macrophage-endothelial 3D co-culture. This data could offer insight into the biological mechanisms of ultrasound which would ultimately help identify novel disease applications where ultrasound may be a viable therapeutic.

Acknowledgements and/or 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. Ngo, O., Niemann, E., Gunasekaran, V., Sankar, P., Putterman, M., Lafontant, A., Nadkarni, S., DiMaria-Ghalili, R. A., Neidrauer, M., Zubkov, L., Weingarten, M., Margolis, D. J., & Lewin, P. A. (2019). Development of Low Frequency (20-100 kHz) Clinically Viable
3. Krzyszczyk, P., Schloss, R., Palmer, A., & Berthiaume, F. (2018). The Role of Macrophages in Acute and Chronic Wound Healing and Interventions to Promote Pro-wound Healing Phenotypes. Frontiers in physiology, 9, 419. https://doi.org/10.3389/fphys.2018.00419
4. Yan, L., Wang, J., Cai, X., Liou, Y. C., Shen, H. M., Hao, J., Huang, C., Luo, G., & He, W. (2024). Macrophage plasticity: signaling pathways, tissue repair, and regeneration. MedComm, 5(8), e658. https://doi.org/10.1002/mco2.658
5. Qin, H. C., Luo, Z. W., & Zhu, Y. L. (2022). Physical energy-based ultrasound shifts M1 macrophage differentiation towards M2 state. World journal of stem cells, 14(2), 214–218. https://doi.org/10.4252/wjsc.v14.i2.214