Treatment of Facial Wounds with Botulinum Toxin A Improves Cosmetic Outcome in Primates
Holger G. Gassner, M.D., David A. Sherris, M.D., and Clark C. Otley, M.D.
Surgeons have constantly sought to achieve the most aesthetic scar. A major factor determining the final cosmetic appearance of a cutaneous scar is the tension acting on the wound edges during the healing phase. Since Theodor Kocher pioneered the alignment of skin incisions with Langer's lines in 1892, surgical techniques that at-tempt to overcome closing tension have become standard. Yet, no treatment has been available to minimize under-lying muscle contractions, which are the major cause of this tension. Botulinum toxin A is a potent drug that produces temporary muscular paralysis when injected locally'. It has proven to be safe and effective in the treatment of a variety of disorders, including hyperkinetic facial lines. The objective of this randomized, double-blind, placebo-controlled primate study was to investigate the efficacy of a single injection of botulinum toxin A to improve the cosmetic appearance of cutaneous scars. Symmetric pairs of standardized excisions were performed on either side of the forehead of six primates. The half foreheads were randomized to the botulinum toxin A treatment side versus the placebo injection side. A panel of three blinded facial surgeons assessed the cosmetic appearance of the mature scars 3 months postoperatively. The wounds that had been immobilized with botulinum toxin A were rated as significantly better in appearance than the control wounds (p < 0.01). Histologic examination confirmed that all scars were mature. Blinded, randomized, placebo-controlled human clinical trials are presently under way at the Mayo Clinic. (Plast. Reconstr. Surg. 105: 1948, 2000.)
In 1816, Jules Cloquet reported that contraction of the underlying musculature creates ridges in the skin.' Nearly two decades later, Guillaume Dupuytren observed that circular cutaneous wounds inflicted with a round awl eventuated in linear clefts.2 Using a similar technique, Karl Langer, a Viennese professor of anatomy, created a map of natural cutaneous lines in cadavers that reflect the tension created by underlying muscular contraction.'
The modern concept of the relaxed skin tension lines advances Langer's works. In general, these lines lie perpendicular to the tension vector of the underlying muscular contraction. Scars aligned with them are subject to reduced tension and heal well, whereas scars oriented against relaxed skin tension lines are subject to repetitive tension, resulting in scar hypertrophy. Standard surgical techniques attempt to avoid muscle pull on wounds by aligning them with the lines, undermining the wound edges or creating skin flaps.' A method to minimize rather than resist or avoid this muscle pull would be a desirable therapeutic modality in the treatment of cutaneous wounds.
Botulinum toxin A (Botox, Allergan, Irvine , Calif. ) is a potent neurotoxin that produces a flaccid paralysis of striated muscle for a period of 2 to 6 months. For more than 30 years, its application has proven safe and efficient in the treatment of a variety of disorders, including hyperfunctional facial lines.' We hypothesized that local botulinum toxin A-induced paralysis of the musculature subjacent to a cutaneous defect would minimize the repetitive tensile forces on the wound edges, resulting in superior cosmetic outcome in the resultant scar. This study was thus designed to investigate the safety and efficacy of botulinum toxin A for temporary pharmacologic immobilization of a healing facial wound in a primate model.
MATERIALS AND METHODS
To closely mimic the effects of muscle activity on human facial skin wounds, the use of an appropriate animal model was mandatory.
Front the Department of Otorhinolaryngology at the Mayo Clinic. Received for publication June 16, 1999: reused September 23, 1999.
Owing to extensive skin laxity and inadequate mimetic musculature, established models like rats, pigs, and horses were not ideal for this purpose. Cynomolgus macaque monkeys (species: Macaca fascicularis) were chosen as a model, because the anatomy of their facial musculature, skin, and skeleton remarkably resembles that of humans.
The study was approved by the Institutional Committee of Animal Care and Use at the Mayo Clinic, and the animals were housed, cared for, and fed in compliance with institutional guidelines. No animal was killed. All procedures were accomplished with anesthesia consisting of ketamine at 20 mg/kg intramuscularly (Ketaset, Fort Dodge , Ind. ), xylazine at 0.5 mg/kg intramuscularly (Rompun, Bayer, West Haven , Conn. ), and isoflurane at 1% (Abbott, Abbott Park , Ill. ).
Because the frontalis, procerus, and corrugator supercilii muscles constantly exert tension on the forehead skin, and because paralysis of these muscles would lead to no functional deficit, the forehead was chosen for the excision site. To minimize local variables, the experimental and control excisions were each planned in symmetric anatomic location in the same individual animal. Three Y-shaped excisions with their main axis perpendicular to the relaxed skin tension lines were planned symmetrically in relation to the midline on each side of the forehead (Fig. 1). Because maximal precision was mandatory, a template was used to determine the location and outline of the excisions. An experienced facial plastic surgeon (D.A.S.), blinded to the experimental conditions, performed all excisions. Using standard surgical technique, the skin and sub-cutaneous tissue were excised, and the frontalis muscle was preserved in the base of the defects.
Subsequently, one side of the forehead was randomly determined as experimental, and the mimetic musculature adjacent to each excision on that side was injected under direct vision with 7 units of botulinum toxin A in 0.9% saline (25 units/ml). This resulted in a total dose of 21 units of botulinum toxin A per half forehead. The control side was injected in the same fashion with an equal volume of 0.9% saline alone. Subsequently, all of the wounds were closed in the same fashion with a single 6-0 chromic gut buried suture (Ethicon, Titusville, N .J.) and multiple 5-0 black monofilament nylon superficial sutures (Ethilon, Ethicon).
Three experienced facial surgeons, who were not present during the surgical procedures, were used as blinded observers to evaluate the cosmetic appearance of the scars at 1, 4, and 12 weeks postoperatively. They were asked to evaluate the width, elevation, and color match of the scars with the surrounding skin to judge the overall cosmetic appearance. Care was taken to sedate the animals deeply for each assessment so the evaluators were not able to recognize the paralyzed side of the fore-head. First, the evaluators were asked to score each scar on a 10-cm visual analog scale. Second, the observers were asked to rate each scar as better than, equal to, or worse than its symmetric counterpart. Finally, the assessors were asked to examine the groups of three scars on either side of each animal's forehead. They were asked to determine that both groups of scars had healed equally or that one group of scars had healed with a cosmetically more favorable result than the group on the contralateral side.
Twelve weeks postoperatively, representative sections of the scars were excised using a 4-mm punch. The biopsy specimens were embedded in formalin, cut into 25-um-thick sections, and stained with hematoxylin and eosin for evaluation.
From the third day postoperatively, marked paralysis of the botulinum toxin A-treated side was observed in all six animals. Extraocular muscle movement and eyelid closure were not compromised. No evidence of paralysis in the foreheads of any of the animals was noted at 12 weeks postoperatively.
At 1 and 4 weeks postoperatively, none of the blinded ratings revealed a significantly better cosmetic appearance than the experimental or the control wounds.
The 36 forehead scars (three experimental scars and three control scars per animal) were evaluated on a 10-cm analog scale by each assessor independently. The mean ratings of the three assessors at 12 weeks postoperatively reached a higher score on the experimental side in 16 of 18 of the symmetric pairs of scars (Fig. 2). The mean scores by assessor no. 1 were 9.4 for the experimental scars and 8.1 for the control scars; those by assessor no. 2 were 8.0 for the experimental scars and 7.3 for the control scars; and those by assessor no. 3 were 7.9 for the experimental scars and 7.3 for the control scars. The mean scores across the three assessors were 8.4 (SD 1.0) for the experimental side and 7.6 (SD 0.9) for the control side. The statistical assessment of an intervention effect was based on using the average rating across the three raters and fitting a two-factor (intervention, site) repeated measures analysis of variance model, taking into account the correlation of measurements obtained on the same animal. Based on this analysis, the scars on the experimental side were rated significantly better than the scars on the control side (p < 0.01). Figure 3 depicts one representative pair of an experimental and a control scar.
At 12 weeks postoperatively, the three assessors were asked to examine the groups of three scars on either side of each animal's forehead. The assessors could then determine that both groups of scars had healed equally or that one group of scars had healed with a cosmetically more favorable result than the group on the contralateral side. A consensus score was de-rived from the majority of the votes. By this consensus score, the experimental sides were assessed as better than the control sides in all six animals. Based on the two-tailed one-sample binominal test, this result is statistically significant (p < 0.031) (Table I).
FIG. 2. Three blinded observers rated each scar on a 10-cm visual analog scale (0 points, the worst possible out-come; 10 points, the best possible outcome). The bars display the mean differences of the scores of the paired experimental and control scars across the three observers (12 weeks post-operatively).
At 12 weeks postoperatively, scar tissue in the dermis could be identified by light microscopy in 23 of the 36 samples. The dermal scars, as illustrated in Figure 4, are characterized by collagen fibers in disarray, delimited by a transition zone that is confluent with the more regularly oriented collagen fibers of the uninjured tissue. All identified scars were mature with no sign of inflammation or ongoing re-modeling.
Immobilization is a basic therapeutic principle in wound healing, common to the treatment of lesions of all kinds. Casts, plates, and sutures minimize the negative effects of muscle tension on healing tissues. Because tension is one of the chief factors determining the degree of scar formation, this principle also holds true in skin lesions.12
The carefully planned execution of an elective skin incision frequently achieves the best aesthetic result. Various techniques, such as placing scars in line with the relaxed skin tension lines, using local flaps, or undermining wound edges, are applied to reduce excessive tension on incisions.13 These techniques, how-ever, minimize rather than eliminate the tension acting on the healing wound. Repeated microtrauma, caused by continuous displacement of injured tissue, induces a prolonged inflammatory response and an increased metabolic activity during the healing process. As a consequence, extracellular deposition of collagen and glycosaminoglycans can intensify and lead to hypertrophic scars. Temporary paralysis of the muscle underlying a wound is a new technique to minimize tension on the healing wound edges. The experienced surgeon can achieve such paralysis with local botulinum toxin A injection in a predictable fashion and rare local side effects of' the treatment can include diffusion of the neurotoxin to adjacent muscle groups. This may result in a temporary functional deficit, such as lagophthalmos, Be surgical closure of' cutaneous wounds, lo-cal anesthetic and vasoconstrictive agents, such as I% lidocaine with epinephrine 1:100,000 (1% Xylocaine with epinephrine 1:100,000, Astra Pharmaceuticals), are routinely injected. In our experience, (he extent of' immediate muscular paralysis achieved by the local anesthetic agent is helpful in predicting the extent of paralysis resulting from the neurotoxin. In addition, we believe that the injection of a vasoconstrictive agent helps to control local diffusion of the neurotoxin and this allows a more precise and safer treatment of the various muscle groups.
Blinded Assessment of the Cosmetic Appearance of the
Scars as Better on One Side or as Equal (12 Weeks
No. Assessor I Assessor 2 Assessor 3 Score
1 + + +
2 + ? + +
3 + + - +
4 + + + +
5 + + + +
6 + + + +
+, assessment of the experimental side as better; -, assessment of the experimental side as worse; and assessment of both sides as equal.
thus precisely immobilize the skin adjacent to certain cutaneous wounds. Traumatic wounds are often unfavorably aligned with respect to relaxed skin tension lines. Our primate data demonstrate that wounds riented unfavorably to these lines benefit from immobilization with botulinum toxin A injection. It is reasonable to assume that elective skin incisions also would benefit from pharmacologic immobilization. In addition to the reduction in mechanical distortion of the wound, early immobilization in elective procedures would also allow the surgeon to use finer sutures and thus achieve an improved cosmetic result.
Botulinum toxin A injections are safe and reliable when performed by an experienced surgeon. The dosage required for local immobilization of a facial wound usually does not exceed 1 unit of Botox/kg of body weight. In a previous primate study, no systemic side effects were observed at dosages below 33 units/kg body weight." A disadvantage of botulinum toxin A, however, is the delayed onset of the muscular paralysis. The injecting surgeon has no immediate feedback on the extent of the resulting muscular paralysis. Therefore, the
The anatomy of the facial skeleton and musculature of the cynomolgus closely mimic those of humans. As confirmed by histologic examination, the microanatomy of the primates' skin also greatly resembles that of human skin. In most established animal models, tissue injuries tend to heal more quickly than in humans. A previous study demonstrated that healing skin wounds in the rhesus monkey mature by 2 months. This was also the case in the present study, as all of' the scars at the termination of' the study at 3 months were histologically mature, with no sign of inflammation or ongoing remodeling. The animals used are capable of hypertrophic scarring, as was observed on one of the research animals from an old injury on the posterior cranium. No keloids were observed in the animals. We believe that the present animal data can be translated to human use, as the data were obtained from mature scars and in a homologous model. Blinded, randomized, placebo-controlled human trials of the use of botulinum toxin in human scar formation are presently underway.
The cosmetic appearance of unfavorably oriented cutaneous scars in a primate model was improved by pharmacologically induced immobilization of the surrounding tissue over an extended period of time during the healing phase (p = 0.01).
For the first time, the macaque was used as a model in facial cutaneous wound healing. Be-cause of the homology with human craniofacial anatomy and cutaneous microanatomy, we recommend the Cynomolgus macaque as a model for future studies.
The low side-effect profile of botulinum) toxin A and the efficacy in improving scar appearance in an animal model justify the treatment of selected human skin lesions in con-trolled human clinical trials.
It has not escaped our attention that immobilization of healing tissues is a fundamental therapeutic principle and that the presented treatment may find application in other areas, such as musculoskeletal lesions, as well.
The authors thank Craig S. Frisk. D.V.M.. Ph.D., Michael C. Blanco, D.V.M., and co-workers for the care of the animals; Anthony E. Brissett . M.D., and David R. Byrd. M.D., for performing blinded assessments; Matthew A. Kienstra, M.D.. for editing the manuscript; Michelle 'I'. Franke and Denise H. Rogers for assistance in preparing the manuscript: Christopher D. Marrs for technical assistance; and Amy Weaver for performing the statistical analysis.
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