Understanding the Biological Effects of Non-thermal, Non-cavitational Therapeutic Ultrasound in Chronic Wound Healing
Authors:   , Boerman Olivia , Lewin Peter Andreas , Spiller Kara L 
Published: August 2022

The purpose of this thesis was to elucidate the effects of low-frequency (20 kHz), low-intensity (100 mW/cm²) (LFLI US) therapeutic ultrasound on macrophage behavior and phenotype. Chronic wounds, such as venous and diabetic ulcers, affect approximately 6.5 million patients in the United States alone. Current standard protocols for wound management do not guarantee healing and include weekly re-dressings and debridement, needed on average for 13 months, to maintain a wound environment that is conducive to passive self-healing. Hence, there is a clear clinical need to develop alternative treatments that promote active (as opposed to passive) healing and shorten healing time. It has been previously reported that treatment with LFLI US significantly (p < 0.03) reduces venous ulcer (VU) size in vivo as compared to VUs treated with a sham device. In a recently completed pilot study of diabetic ulcers (DUs), LFLI US treated wounds healed in nearly one third the time of the sham-treated wounds. However, the mechanisms by which LFLI US promotes chronic wound healing are not understood. There is evidence that the cause of impaired healing is the dysregulation of macrophage phenotype, especially the defective transition from pro-inflammatory (M1) to pro-healing (M2) macrophages, which has been identified as a major source of chronic inflammation. Previous characterization of tissue debrided from diabetic ulcer patients treated with ultrasound or a sham device showed a decrease in M1/M2 related genes in healing wounds treated with LFLI US compared to non-healing wounds treated with a sham device over time. Therefore, the purpose of this thesis was to (1) characterize the effects of LFLI US on wound inflammation using debrided human chronic wound tissue, (2) develop and validate an experimental design to reliably conduct in vitro ultrasound experiments eliminating reflections, and (3) elucidate the direct effects of LFLI US on macrophage functional behavior and phenotype. First, debrided human chronic venous ulcer tissue from patients treated with LFLI US or a sham device were collected. RNA was extracted from these samples and processed using a multiplex gene expression analysis tool, Ampliseq. 477 genes were found to be differentially expressed (DEGs) in the ultrasound group when compared to the sham group (p < 0.05; FC >2) and notably, gene set enrichment analysis of these DEGs showed the most enriched gene set from downregulated DEGs was the inflammatory response gene set. Then, an acoustic chamber designed, modeled, and developed for controlled in vitro ultrasound experiments mitigating reflections. FEA modeling showed 36-fold greater than intended acoustic pressure propagated to cells without the use of this developed acoustic chamber underscoring the necessity of accounting for reflections when propagating ultrasound in vitro. Finally, in vitro exposure of M1 macrophages to LFLI US showed a significant upregulation of 20 genes associated with mitochondrial and cell membrane activity and although not significant, a decrease protein secretion of inflammatory cytokines IL6 and TNF-[alpha] in ultrasound treated groups compared to sham treated groups. All together, these results suggest that LFLI US reduces wound inflammation in chronic wound tissue and a possible biological mechanism of this ultrasound-assisted healing may be through the decrease of macrophage secretion of inflammatory cytokines through modulating mitochondrial and cell membrane activity. This project lays the critical foundation necessary to begin exploring the possible biological mechanisms of LFLI US healing. With the understanding of how LFLI US modulates cell behavior and phenotype, ultrasound-based therapeutics have the potential to advance healing in a wide spanning number of diseases and dysfunctions ultimately enhancing patient quality of life.