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

Biomedical Imaging and Instrumentation

Biomedical Imaging and Instrumentation - Poster Session B

Poster G2 - Effects of Lead-Based vs. Lead-Free Low-Frequency, Low-Intensity Ultrasound Devices on VEGF Secretion from Endothelial Cells in a 3D Scaffold

Thursday, October 24, 2024
2:45 PM – 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Research has shown that the application of low-frequency, low-intensity ultrasound (LFLI US) accelerates healing processes within cell tissue (Samuels et al., 2013). In one human study, diabetic ulcers closed approximately 3 times faster when treated with ultrasound (Ngo et al., 2019). Beyond wound healing, LFLI US also holds promising potential in areas such as pain management (Gam et al., 1995) (Gehling et al., 2007), nerve stimulation (Gavrilov et al., 1996), and transdermal drug delivery (Sunny et al., 2012).

Piezoceramics like lead zirconate titanate (PZT) are vital to the function of therapeutic ultrasound devices for their ability to generate ultrasound waves via the piezoelectric effect. Consequently, these devices typically contain over 60% lead. Increasing knowledge about the ramifications of lead-containing appliances on human health and the environment has prompted global regulatory actions like the 2003 European Union law on the “Restriction of Hazardous Substances,” which call for a shift away from lead-based medical devices in the near future (EU, 2003).

To evaluate the therapeutic effectiveness of lead-free piezoceramics and explore viable alternatives to traditional lead-based materials, both lead-based and lead-free devices were manufactured and biological testing on human cells was conducted post-ultrasound application. More specifically, this study monitored the secretion of vascular endothelial growth factor (VEGF), a key stimulator of angiogenesis, in human umbilical vein endothelial cells (HUVECs).

Materials and

Methods:  A flexural cymbal transducer design, optimized for operation between frequencies of 20 and 100 kHz, guided the manufacturing process (Sunny et al., 2012). Devices consisted of two identical convex brass caps adhered to either side of piezoceramic disks (Ferroperm Piezoceramics), as well as a positive and negative lead.

Brass sheets were manually cut, sanded, and shaped. A PZT-based Pz26 disk was used for lead-based devices, while a sodium bismuth titanate (NBT) Pz12 disk was used for the lead-free. Individual components were fixed in place with bonding epoxy, wires were attached with cold solder, and the entire assembly was potted in polyurethane epoxy (Spurr’s Epoxy). The resonant frequency of each device was determined using a vector network analyzer and Teledyne RESON probe.

Four groups of HUVECs were seeded in a 3D collagen scaffold. Ultrasound was applied to each group for 15 minutes at varying intensities: 50 mW/cm², 100 mW/cm², 150 mW/cm², while one group received no treatment. After a 48-hour incubation period, the conditioned media was collected and an enzyme linked immunosorbent assay (ELISA) was used to quantify VEGF secretion following manufacturer’s (PeproTech) protocol. This procedure was repeated with both lead-based and lead-free transducers. The differences between absorbance values of each sample–collected at 450 nanometers and 605 nanometers–were used to create a standard curve with a four-parameter logistic (4PL) line of best fit, and further calculate VEGF concentrations in pg/mL. Results were analyzed for significant (p < 0.05) differences using a one-way ANOVA and Tukey’s post hoc analysis.

Results, Conclusions, and Discussions: There was a significant difference (p < 0.05) in VEGF secretion when comparing the varying ultrasound intensities to the control group using lead-based devices. While ultrasound application at any intensity led to higher VEGF concentration than the baseline set by the control group, intensities of 50 and 100 mW/cm² stimulated more VEGF secretion than 150 mW/cm².

Although not significantly different, we observed that lead-free ultrasound devices stimulate the same concentration of VEGF secretion in the 100 mW/cm² group when compared to the control group. This data suggests that applying therapeutic ultrasound at 50 mW/cm² and 100 mW/cm² is potentially more beneficial than application at 150 mW/cm² in the context of wound healing; HUVECs displayed the greatest increase in VEGF concentration at these intensities with lead-based applicators. In turn, this would lead to increased angiogenesis.

Additionally, these results convey that NBT may be a successful substitute for PZT in ultrasound-generating piezoceramics. It is necessary to repeat a 48-hour ELISA with lead-free applicators to validate this finding, yet lead-free devices nonetheless appear to reproduce similar biological effects in HUVECs as lead-based devices.

Acknowledgements (Optional):