Diabetic db/db mice (BKS.Cg-Dock7m +/+ Leprdb/J) were obtained from the Jackson Laboratory. Experiments were performed on 12–16 week-old male mice. Only mice with fasting blood glucose >250 mg/dl were included in the study. Mice were housed under environmentally controlled conditions, with a 12-h light/dark cycle. Water and food were available ad libitum. To minimize bias, mice were randomly assigned to experimental groups, and the resulting samples were coded and analyzed in a blinded fashion. For all experimental procedures, mice were anesthetized with isoflurane (3% for induction and 2% for maintenance). For euthanasia, mice were anesthetized with 3% isoflurane, followed by cervical dislocation. All procedures involving animals were approved by the Animal Care Committee at the Jesse Brown Veterans’ Affairs Medical Center.

Mice were subjected to excisional wounding, as described previously (Weinheimer-Haus et al., 2014; Roberts et al., 2021a; Roberts et al., 2023). In brief, mice were anesthetized using isoflurane, and their dorsum was shaved and cleaned with alcohol. Four 8-mm wounds, spaced ∼5 mm apart, were made on the back of each mouse using a dermal biopsy punch and covered with Tegaderm (3M, Minneapolis, MN) to keep the wounds moist and maintain consistency with the treatment of human wounds. Wound closure was assessed for all four wounds from each of three mice (N = 12 total). Two wounds from each of three mice were analyzed for each histological and growth factor assay (N = 6 total for each assay).

All mice receiving local LIV treatment were anesthetized using isoflurane and then placed on their backs on the experimental platform (Figure 1). For the first set of experiments, local LIV was applied near the wounds via an indenter driven by an oscillating motor. Mice were randomly assigned to groups receiving one of two LIV protocols based on our previous studies on whole-body LIV (Weinheimer-Haus et al., 2014; Roberts et al., 2021a); the first involved application of LIV with low frequency (45 Hz) and acceleration (0.3 g) (LL), and the second involved high frequency (90 Hz) and acceleration (1.0 g) (HH). Accelerations were measured using an accelerometer placed on the tip of the indenter, and inputs to the oscillating motor were controlled using custom software. Each group was further split into two subgroups, with one subgroup receiving LIV daily and the second subgroup receiving LIV every other day. For mice that received LIV daily, treatment started on the day of injury (day 0) and ended on day 9. For mice that received LIV every other day, treatment occurred on the day of injury (day 0), day 2, day 4, day 6, and day 8. Mice were euthanized, and wounds were harvested on day 10. Control mice were anesthetized and placed in the experimental setup but did not receive LIV treatment.

For the next set of experiments, local LIV was delivered near the wounds via piezoelectric disks (Figure 2). After induction of anesthesia, piezoelectric disks were placed near the wounds, and input parameters were adjusted to deliver vibrations with a frequency of 90 Hz and an acceleration of 0.02 g; these were the largest accelerations that could be generated using these disks, likely due to the small mass and vibration characteristics of the disks. Accelerations were measured near the wound site using an accelerometer. Experimental mice received LIV daily, starting from the day of injury (day 0) and ending on day 9. Mice were euthanized, and wounds were harvested on day 10. Control mice were anesthetized and placed in the experimental setup but did not receive LIV treatment.

Wound closure was assessed in digital images of the external wound surface taken immediately after injury and on days 2, 6, and 10 post-injury. The wound area was measured using Fiji Image J and expressed as a percentage of the area immediately after injury.

Re-epithelialization and granulation tissue thickness were measured in cryo-sections taken from the center of the wound (found by serial sectioning through the entire wound) and stained with hematoxylin and eosin (Weinheimer-Haus et al., 2014; Roberts et al., 2021a; Roberts et al., 2023). Digital images were obtained using a Keyence BZ-X710 All-in-One Fluorescence Microscope (Keyence, Itasca, IL, United States), with a ×2 or ×20 objective, and analyzed using ImageJ software. The percentage of re-epithelialization, length of epithelial tongues, and granulation tissue area were measured in three sections per wound and averaged across sections to provide a representative value for each wound.

Angiogenesis was assessed by immunohistochemical staining for CD31 (390, 1:100; BioLegend, San Diego, CA, United States), macrophage accumulation by staining for F4/80 (BM8, 1:100, Thermo Fisher, Waltham, MA, United States), and neutrophil accumulation by staining for Ly6G (1A8, 1:100, BD Pharmingen, Franklin Lakes, NJ, United States). Collagen deposition was assessed using Masson trichrome staining (IMEB, San Marcos, CA, United States). For each assay, digital images were obtained covering the wound bed (2–3 fields using a ×20 objective), and the stained percentage area was quantified by the number of clearly stained pixels above a threshold intensity and normalized to the total number of pixels. The software application allowed the observer to exclude artifacts. For each assay, three sections per wound were analyzed.

Wounds that had been snap-frozen in liquid nitrogen and stored at −80 C were homogenized in cold PBS (10 μl of PBS per mg of wound tissue) supplemented with a protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, United States) using a Dounce homogenizer and then centrifuged. Supernatants were used for enzyme-linked immunoassays of IGF1 and VEGF (R&D Systems, Minneapolis, MN, United States).

Values are presented as the means ± standard deviation. For oscillating motor results, measurements of wound healing or wound growth factors were compared between low- and high-intensity LIV, daily and every-other-day, and sham control treatment groups using two-way ANOVA. Sidak’s multiple-comparisons test was used when ANOVAs demonstrated significance. For piezoelectric disk results, measurements of wound healing or wound growth factors were compared between LIV and sham treatment groups using t-tests. Differences between groups were considered significant if p ≤ 0.05.

Diabetic db/db mice were treated with one of four LIV protocols: low frequency with low acceleration (LL) or high frequency with high acceleration (HH), applied either daily or every other day. Over the course of the experiment, mice lost ∼5% body weight, which was similar across protocols, and none of the protocols altered blood glucose levels (Table 1). All the LIV protocols accelerated wound closure on day 10 post-injury, as assessed by external measurements, compared to sham controls (Figure 2). Similarly, all the LIV protocols increased histological measurements of re-epithelialization on day 10 post-injury compared to the sham control (Figure 2). There were little, if any, differences in the effects of the different LIV protocols on these measurements of epithelial closure. In short, locally applied LIV enhances wound closure in diabetic mice, independent of vibration intensity within the range tested in these experiments and of whether LIV was applied daily or every other day.

Similarly, all the LIV protocols increased the granulation tissue area (Figure 3, example images in Figure 2) and enhanced angiogenesis assessed by CD31 staining (Figure 3), on day 10 post-injury compared to sham controls. Again, there were little, if any, differences in the effects of the different LIV protocols on these measurements of dermal healing. In short, locally applied LIV enhances angiogenesis and granulation tissue formation in wounds of diabetic mice, independent of vibration intensity within the range tested and of whether LIV was applied daily or every other day.

Our previous studies on whole-body LIV showed that LIV-enhanced wound healing was associated with increased wound IGF1 levels in obese and diabetic db/db mice (Weinheimer-Haus et al., 2014; Roberts et al., 2021a). Importantly, knockdown of liver IGF1 blunted the whole-body LIV-induced increase in wound IGF1 levels and improved healing in wounds of high-fat diet-fed obese and insulin-resistant mice (Roberts et al., 2023). In contrast to these studies, locally applied LIV only increased wound IGF1 levels when applied daily at high intensity; the other LIV protocols did not increase IGF1 levels, despite enhancing wound healing (Figure 4). In addition, local LIV induced an intensity-dependent increase in wound VEGF levels whether applied daily or every other day (Figure 4). Therefore, although specific protocols of locally applied LIV increased IGF1 and VEGF levels, other factors likely contribute to local LIV-enhanced wound healing, as shown in Figures 2, 3.

To advance our studies toward a wearable device, diabetic mice were treated daily with LIV delivered by piezoelectric disks at a frequency of 90 Hz and an acceleration of 0.02 g. This was the largest acceleration achievable with these disks. As with the oscillating motor experiments, mice lost <5% body weight, and blood glucose levels were not altered over the course of the experiment (Table 2). LIV delivered by piezoelectric disks showed a trend of accelerating wound closure on day 10 post-injury, as assessed by external measurements, compared to sham controls (Figure 5). Similarly, LIV delivered by piezoelectric disks showed a trend of increasing histological measurements of re-epithelialization compared to sham controls (Figure 5). In short, LIV delivered by piezoelectric disks shows potential for enhancing wound closure, although the small accelerations delivered by this method may have limited the efficacy of treatment.

Importantly, LIV delivered by piezoelectric disks significantly increased the granulation tissue area on day 10 post-injury (Figure 6, example images in Figure 5) and increased angiogenesis assessed by CD31 staining (Figure 6) compared to sham controls. Therefore, local LIV delivered by piezoelectric disks appears to have a stronger impact on dermal healing than epidermal healing of wounds in diabetic mice.

In contrast to our previous studies on whole-body LIV (Weinheimer-Haus et al., 2014; Roberts et al., 2021a) and our experiments with local LIV applied using an oscillating motor (Figure 4), locally applied LIV delivered by piezoelectric disks did not increase wound IGF1 levels (Figure 7). In contrast, LIV delivered by piezoelectric disks markedly increased wound VEGF levels (Figure 7). Therefore, the production of VEGF in wounds appears to be more sensitive to local LIV signals than IGF1.