Noncontact, low-frequency ultrasound therapy enhances neovascularization and wound healing in diabetic mice

Abstract

Background: Chronic wounds are a major source of morbidity for patients and represent a significant health burden. Implementing noninvasive techniques that accelerate healing of these wounds would provide great benefit. Ultrasound appears to be an effective modality for the treatment of chronic wounds in humans. MIST Therapy is a noncontact, low-frequency ultrasound treatment delivered through a saline mist. A variety of mechanisms have been proposed to explain the efficacy of ultrasound therapy, but the underlying molecular and cellular pathways impacted by this technique remain unclear. The in vivo effect of noncontact, low-frequency ultrasound was therefore examined in a humanized excisional wound model.

Methods: The treatment group received noncontact, low-frequency ultrasound therapy three times per week, whereas the control group received a standard dressing change. Wounds were photographed at regular intervals to calculate healing kinetics. Wound tissue was harvested and processed for histology, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay.

Results: The MIST group demonstrated significantly accelerated wound healing, with 17.3 days to wound closure compared with 24 days in the controls (p < 0.05). This improvement became evident by day 9, with healing evidenced by significantly decreased mean wound area relative to original size (68 percent versus 80 percent; p < 0.01). Expression of markers of neovascularization (stromal cell-derived factor 1, vascular endothelial growth factor, and CD31) was also increased in the wound beds of noncontact, low-frequency ultrasound-treated mice compared with controls.

Conclusion: Noncontact, low-frequency ultrasound treatment improves neovascularization and wound closure rates in excisional wounds for diabetic mice, likely because of the stimulated release of angiogenic factors.

Figures

Fig. 1

Representative images at time intervals demonstrating the (above) increased rate of wound closure and increased vascularity of the wound bed in the noncontact, low-frequency ultrasound (NLFU) group. (Below, left) Wound healing kinetics demonstrating significantly decreased wound size in the noncontact, low-frequency ultrasound group relative to controls starting at day 9 and (below, right) shorter time to wound closure in the noncontact, low-frequency ultrasound group (*p < 0.05).

Fig. 2

Histology of noncontact, low-frequency ultrasound–treated and control wounds. Black vertical dotted lines identify the original wound margin. (Above, left) Hematoxylin and eosin stain demonstrating a more organized architecture and (above, right) increased dermal thickness and cutaneous integrity in noncontact, low-frequency ultrasound treatment group with (below) significantly increased collagen density on Masson trichrome and picrosirius red staining. Scale bar = 100 μm (*p < 0.05). NLFU, noncontact, low-frequency ultrasound.

Fig. 3

Immunohistochemistry and quantitative polymerase chain reaction for neovascularization in noncontact, low-frequency ultrasound–treated and control wounds. (Above, left, and center) Increased VEGF mRNA and protein levels in the noncontact, low-frequency ultrasound group, (above, right, and below) with subsequently increased CD31 mRNA and protein levels. VEGF, vascular endothelial growth factor; NLFU, noncontact, low-frequency ultrasound. Scale bar =100 μm (*p < 0.05).

Fig. 4

SDF-1 regulation in noncontact, low-frequency ultrasound–treated and control wounds. SDF-1 mRNA levels, protein concentration, and immunofluorescent protein expression were significantly enhanced by noncontact, low-frequency ultrasound treatment. Scale bar = 100 μm (*p < 0.05).