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Page 6 of 14 Nakamoto et al. Plast Aesthet Res 2024;11:54 https://dx.doi.org/10.20517/2347-9264.2024.82

COL3a1, ACTA2, and MMP1, with the abundance of each target normalized to EEF1A1 [Table 1]. The data
shown for each target are the ∆∆Ct values. In each wound, the relative expression of each gene was
determined with EEF1A1 as the housekeeping gene∆.

Statistical analysis
Values are expressed as means ± standard error of the mean (SEM). All analyses were performed with
GraphPad Prism 10 (GraphPad Software, La Jolla, CA) software. Statistical significance was set at P < 0.05.

The differences in epithelialization rate on Day 14 between groups were compared using a paired t-test.

The time from the skin graft to when the epithelialization rate exceeded a certain percentage (85%, 90%, and
95%) was compared using survival time analysis with the log-rank test.

The blood flow data from the PeriCam at each time point were compared using two-way ANOVA with
repeated measures, followed by Bonferroni test.

The mean values for epidermal thickness and the numbers of neutrophils, macrophages, and blood vessels
were calculated for each wound. Then, these values were compared using paired t-test.

The relative gene expression levels in each wound were compared by paired t-test.

RESULTS
Wounds epithelialization results
All wounds had no complications such as infection or hematoma [Figure 4]. The epithelialization rate on
Day 14 in the beta-blocker group (84.46% ± 5.893%) was significantly higher than that in the control group
(74.30% ± 5.861%) (P = 0.0158) [Figure 5]. The days when the epithelialization rate exceeded 85%, 90%, and
95% in the beta-blocker group were significantly shorter than those in the control group (P = 0.0354, P =
0.0104, and P = 0.0313, respectively) [Figure 6]. There were no significant differences in the wound blood
flow between the groups at any measured time point [Figure 7]. There were no hypoglycemia [Table 2] and
hemodynamic changes, i.e., hypotension [Table 3] and bradycardia [Table 4]. These results indicate that
topical application of timolol significantly promotes epithelialization of mesh skin grafted full-thickness
burn wounds through mechanism(s) other than improving wound blood flow.

Histological results
The thickness of the epithelial layer in the treatment group was significantly thicker than those in the
control group (213.1 ± 15.64 µm in the treatment group vs. 183.5 ± 16.25 µm in the control group, P =
0.0203) [Figure 8]. This result supports the finding that the epithelialization rate on Day 14 in the treatment
group was significantly higher. There were no significant differences in the numbers of neutrophils,
macrophages, and blood vessels per field between the treatment and control groups [Figure 9]. The number
of neutrophils was 28.88 ± 9.864 in the treatment group and 40.61 ± 10.95 in the control group (P = 0.2433).
The number of macrophages was 1.492 ± 0.269 in the treatment group and 1.733 ± 0.4517 in the control
group (P = 0.5763). The number of blood vessels was 2.817 ± 0.4156 in the treatment group and 3.475 ±
0.6433 in the control group (P = 0.2688). These results indicate that timolol’s positive effects on wound
epithelialization are not linked to the severity of inflammation and angiogenesis in the wounds.

RNA abundance measurements
There were no significant differences in the levels of RNA abundance relative to EEF1A1 for several