The effect of botulinum toxin injection dose on the appearance of surgical scar

Early postoperative injection of botulinum toxin type A (BTxA) can reduce surgical scar hypertrophy. BTxA injection at different time points is associated with different levels of efficacy, but the efficacy of different doses of BTxA for scar management has not investigated. The purpose of this study was to investigate the effect of different doses of BTxA administered early after surgery on scar improvement through a split-scar experiment. The study included 22 patients who underwent surgery between September 2019 and October 2020. High- and low-dose BTxA was randomly administered into each half of the surgical wound closure immediately after surgery. One half of the incision was injected with a low dose (4 U) of BTxA, and the other half was injected with a high dose (8 U). The scars were then evaluated at postoperative 6 months using the modified Stony Brook Scar Evaluation Scale (mSBSES), and patient satisfaction was evaluated using the Visual Analogue Scale (VAS). The occurrence of complications or adverse events was also recorded. Twenty patients completed the study and were analyzed. Compared with the low-dose sides, the high-dose sides had significantly better mSBSES scores and significantly higher VAS scores (p < 0.01, respectively). No serious adverse reactions or post-injection complications were observed. Immediately after the operation, high-dose BTxA (that is within the therapeutic range) injection improved the appearance of postoperative scar more than low-dose injection.


Scientific Reports
| (2021) 11:13670 | https://doi.org/10.1038/s41598-021-93203-x www.nature.com/scientificreports/ Registration date was 05/01/2021, Registration number was ChiCTR2100041766) (www. chictr. org. cn). All participants were informed about the study through a clear and simple written description of the procedure to ensure their understanding, and they provided their informed consent for participation. Randomized Controlled Trial flow diagram was shown in Fig. 1.
Sample size. The sample size was calculated based on the method described in previously published literature in 2018 15 : An increase of 1 in the VAS score after treatment was considered as clinically significant 15 . Based on this criterion, approximately 18 wounds per group would be necessary to provide a result of real significance with the same standard deviation considering a standard type 1 α error of 0.05 and power of 0.8. Assuming a 10% noncompliance rate for follow-up (evaluation), the required sample size was determined as 20 patients.
The study included participants aged 18 years and above who were scheduled to undergo superficial mass excision (skin type of the patients: Fitzpatrick skin type III or IV) at the Department of Plastic Surgery, First Affiliated Hospital of Zhengzhou University, between September 2019 and October 2020.
The exclusion criteria were (1) known BTxA or albumin allergy, (2) BTxA injection within the past 6 months, (3) a history of neuromuscular disorders, (4) a history of hypertrophic scars and keloids, and (5) current pregnancy or breast feeding.
Procedure. Z. C. generated the random allocation sequence, enrolled and assigned participants. A random number generator was used to generate ones and twos (each time a random number was generated, the maximum value was 2 and the minimum value was 1), which designated left and right, respectively, or upper and lower, respectively. Accordingly, half part of individual participant's wound was randomly assigned to one  After tumor resection, incision tension was assessed. Vertical mattress suture was performed on the subcutaneous layer and dermis with 3-0 to 5-0 absorbable sutures, respectively. After closing the incision, discontinuous epidermal suture was performed with 6-0 or 7-0 nylon sutures. After skin closure, the wound was marked at the midline point. The postoperative wound was treated, with each side randomized to receive either a low dose of BTxA (4 U at every point, with an interval of 1 cm) or a high dose of BTxA (8 U at every point, with an interval of 1 cm) injected intradermally from a site 5 mm away from the wound edges. Patients, injector (Z.R. W.) and scar evaluators (R. P. and H. Z.) were blinded to the dose received.
After operation, the patient was observed in the recovery room for 30 min and the discomfort and adverse reactions (if any) were recorded. Disinfect and replace excipients every 2-3 days according to the surgical site and wound healing. The time of suture removal depends on the site of operation. No additional anti-scar therapy, such as stress therapy or silicone application, was given after surgery. The occurrence of complications or other discomfort was also recorded during the follow-up.
Evaluation of clinical effect. The cohort was observed over a 6-month postoperative follow-up period. At each of the follow-up visits, two plastic surgeons (R. P. and H. Z.) assessed and scored the left and right sides of the incision independently using the mSBSES (The mSBSES contains four subcategories, as shown in Table 1) and VAS (from 0 to 10: 0 = worst and 10 = best) and took standardized digital photographs of the scars 14,15 . The differences in mSBSES score and VAS score between the two groups was used as the primary outcome, and the occurrence of complications or adverse events was considered as a secondary outcome 16 . The patients were required to report adverse events. Throughout the study period, the patients were blinded to the BTxA dose administered to each side. Statistical analysis. IBM SPSS Version 22 (IBM Corp., Armonk, N.Y.) was used to analyze all data. Paired t tests were used to compare the summative scores for VAS, SBSES and its sub-items for each half of the scars. Non-parametric rank sum test was used for measurement data that did not conform to normal distribution (Shapiro-Wilk test was used for normality test, and Wilcoxon test was used for nonparametric test). Statistical significance was accepted at p < 0.05.

Color (redness)
Skin prominently more red than the surrounding skin 0 Scar more red than the surrounding skin 1 Scar of the same color or lighter than surrounding skin 2

Incision line
Prominent incision line 0  Table 2). Patient satisfaction was evaluated using VAS. At the 6-month follow-up, higher satisfaction was reported for the high-dose injection side, and the mean VAS score for the high-dose side was 7.85 ± 1.27 and the low-dose side was 5.20 ± 1.40, with a statistically significant difference between the two sides (p < 0.01).

Discussion
The present split-scar study explores the impact of immediate postoperative administration of high and low doses of BTxA around the area of incision on postoperative scar prevention. In our study, the high-dose and low-dose injection sides showed significant differences, with the high BTxA dose exhibiting better results. With regard to the modified SBSES subcategories, height and color (redness) were not significantly different between the two sides, but the high-dose injection had significantly better results in terms of the other two subcategories, namely, width and incision visibility line. In contrast, Kim et al. used a split-scar study to compare BTxA and non-BTxA injection sides and observed a significant effect in all four modified SBSES subcategories 14 . This difference could be explained by the small sample size of our study. What needs illustration is that the tumors of the patients in this study generally did not invade the muscle, so the muscle tissue remained intact after resection (no muscular tension). After intradermal injection of botulinum toxin, the high-dose group showed better scar appearance. This suggests that the role of botulinum toxin in preventing scar formation is more dependent on non-neuromuscular pathways than on the relaxation of muscle. A series of studies by Xiao et al 10,[17][18][19] systematically elucidate the main mechanisms of botulinum toxin preventing scar formation: (i) Botulinum toxin can inhibit the proliferation of fibroblasts, promote their apoptosis, and inhibit their differentiation into myofibroblasts; (ii) Botulinum toxin can inhibit the expression and secretion of fibrosis related factors. (iii) At the same time, botulinum toxin can change collagen deposition and induce collagen remodeling to some extent while reducing tension. To be specific, Botulinum toxin type A can work as a downstream regulator of TGF-β1 secreted by macrophages, which can reduce the expression of connective tissue growth factor. And botulinum toxin type A acts on the TGF-β1/Smad pathway at the molecular level, thereby inhibiting Scars formed by fibroblasts 11,18 . Gassner et al. described a mean dose of 30 U for 2-to 4-cm forehead wounds in a 2006 report 12 . The reported dose varies from 2.5 to 10 U for each 1-cm scar in different studies 12,20 and most studies use a dose of 5 U for each 1-cm scar to investigate the effect of BTxA on postoperative scar management 4,21 . Therefore, in this study, 4 U was used as the low dose and 8 U as the high dose for each 1-cm scar. Additionally, the wounds were divided into high-and low-dose sides, and this greatly reduced the interference of unrelated confounding factors. The highdose botulinum toxin was found to be superior to the low-dose toxin in terms of reducing the tension around the incision and inhibiting scar hypertrophy. In agreement with these findings, clear differences have been reported in the effects of different doses of BTxA on crow's feet, primary palmar hyperhidrosis, and gummy smile [22][23][24][25] .
The difference in the effect of different doses of BTxA may be related to differences in the diffusion rate of various doses 26 . A study reported that a gradient of denervation occurred throughout the entire muscle with no apparent endpoint when BTxA was administered at doses of 5-10 IU, and both the magnitude of denervation and the extent of the gradient were dose dependent 27 . Therefore, different doses of BTxA may have different effects on the muscles at the injection site. The results of a dose-ranging, electroneurographic study investigating the dose equivalence and diffusion characteristics of BTxA in 2008 showed significant and similar reductions in compound muscle action potential amplitude in the extensor digitorum brevis 2 weeks after injection, with the effects lasting for 12 weeks. Further, the reduction in amplitude increased with increasing doses and with increasing concentration 27,28 . Thus, the dose-dependent effects observed in this study could be explained by these muscle-related mechanisms of BTxA. According to these findings, many other studies have also shown that for botulinum toxin, its volume, dose, and accuracy have the greatest impact on clinical outcomes [29][30][31] .
The main limitation of our study is the small sample size. Therefore, studies with a larger sample size are necessary to confirm the results. Second, it will be important to perform studies to investigate a range of BTxA www.nature.com/scientificreports/ doses within the therapeutic dose in order to determine whether the BTxA dose is directly proportional to the effectiveness of scar prevention.

Conclusion
In the present study, the results show that the high dose of BTxA was more effective than the low dose in the management of scar hypertrophy. These findings indicate that early postoperative high-dose BTxA injections can provide better cosmetic effects than low-dose injections. Therefore, we recommend a high-dose BTxA injection immediately after the procedure to achieve a better scar beautification effect.