Preventive, Cumulative Effects of Botulinum Toxin Type A

in Facial Aesthetics
Shannon Humphrey, MD,*† Birgitte Jacky, PhD,‡ and Conor J. Gallagher, PhDx

BACKGROUND Botulinum toxin Type A (BoNTA) is the gold standard for the treatment of dynamic rhytides
in the face. Recently, clinical observation suggests that individuals who receive regular injections of BoNTA
experience ongoing wrinkle reduction and improvements in overall skin quality not observed in those treated
sporadically.

OBJECTIVE To review scientific evidence of qualitative changes in the skin and the possibility of indirect or
direct effects on fibroblasts affecting fibroblast activity, including collagen production, after repeated treatment
with BoNTA.

MATERIALS AND METHODS We examined the literature for supporting evidence of the effect of repeated
treatment cycles on wrinkle reduction and skin quality; histological changes in collagen structure; alterations in
biomechanical features of the skin; and potential fibroblastic response.

RESULTS Apparent cumulative improvement on wrinkle reduction and additional skin quality attributes
with regular BoNTA treatments suggests an ongoing process of dermal repair. Clinical observation suggests
that BoNTA injections stimulate collagen production and lead to a reorganization of the collagen network
within the extracellular matrix, which in turn may produce improvements in features associated with more
youthful skin. Moreover, evidence suggests that BoNTA may have a direct or indirect effect on fibroblast
activity.

CONCLUSION Clinical observation of progressive wrinkle reduction and qualitative improvements in

a number of skin attributes that accumulate with more frequent injections of BoNTA suggest an ongoing
process of repair leading to prolonged and cumulative effects.

Editorial Assistance paid for by Allergan. S. Humphrey is a speaker, consultant and investigator for Allergan,
Revance, Galderma, Merz, and Evolus. The remaining authors have indicated no significant interest with
commercial supporters.

M edical breakthroughs often come from a single

leap of faith, a clinical observation in the
absence of definitive evidence. In 1992, Carruthers
and Carruthers published their seminal paper noting
the cosmetic effect of botulinum toxin Type A
(BoNTA) on glabellar lines after injection for the
treatment of blepharospasm,1 an observation that
would change the face of cosmetic dermatology and
spur an intense period of study. The neurotoxin has
since become the most requested noninvasive cosmetic
procedure in the United States and is the gold standard
for treatment of dynamic rhytides in the face.2
However, the toxin’s effects may go beyond its
intended short-term outcome on muscular activity.
Mounting evidence demonstrates progressive,
cumulative improvements in resting rhytides and skin
quality after regular, repeated treatments with BoNTA
suggesting a cellular influence in addition to muscle
relaxation. In order to better understand these
apparent results—to make the leap from observation
to possible mechanism of action—we looked for
scientific evidence of qualitative changes in the skin
and direct or indirect effects on a dermal and cellular
level after treatment with BoNTA (Table 1).

*Carruthers & Humphrey Cosmetic Dermatology, Vancouver, British Columbia, Canada; †Department of Dermatology,
University of British Columbia, Vancouver, British Columbia, Canada; ‡Research and External Scientific Innovation,
Allergan plc, Irvine, California; xMedical Affairs, Allergan plc, Irvine, California

© 2017 by the American Society for Dermatologic Surgery, Inc. Published by Wolters Kluwer Health, Inc. All rights reserved.
· ·
ISSN: 1076-0512 Dermatol Surg 2017;43:S244–S251 DOI: 10.1097/DSS.0000000000001404

S244

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 HUMPHREY ET AL

TABLE 1. Evidence of Dermal and Cellular Changes After Treatment With BoNTA

Observation Evidence References

Reduction in sebum production and
improvements in oiliness and pore
size
Increased levels of Procollagen II
Reorganization of collagen fibers
Improvements in elastic recoil and
mean pliability
Improvements in biophysical
parameters
Increased production of PIP,
decreased expression of MMPs, and
increased levels of proalpha
collagen chains
Increased expression of Collagen I
and III and decreased secretion of
MMPs in UVB-irradiated dermal
fibroblasts
Differentially affects disease (Keloid
and HS) vs normal fibroblasts
Clinical measurable observations
Clinical measurable observations
Clinical measurable observations
Clinical measurements
Clinical measurements
In vitro, explant human fibroblasts
In vitro, explant human fibroblasts
In vitro and clinical
Shah, 2008, Rose and Goldberg,
2013, Li and colleagues, 2013,
Min and colleagues, 2015
Chang and colleagues, 2008
El-Domyati and colleagues,
2015
Bonaparte and Ellis, 2014, 2015
Zhu and colleagues, 2017
Oh and colleagues, 2012
Permatasari and colleagues,
2014
Zhibo and Miaobo, 2008, 2009,
Xiao and colleagues, 2009,
2011, Xiaoxue and colleagues,
2014, Jeong and colleagues,
2015

BoNTA, botulinum toxin type A; HS, hidradenitis suppurtiva; MMPs, matrix metalloproteinases; PIP, procollagen type I carboxy-terminal
peptide; UVB, ultraviolet B.

The View From the Bedside
Clinical experience tells us that repeated treatment
with BoNTA produces improvements in skin quality
that have yet to be clearly defined: individuals who
consistently visit the clinic for regular, repeated injec-
tions of BoNTA simply look better than those who do
not, and look better over time, with skin that appears
more youthful and radiant. It is an observation that
begs further investigation and a better understanding
of what constitutes regular treatment (e.g., how many
injections over what period of time).
The effect of regular treatment with BoNTA every few
months over 2 or more years has been investigated in the
literature, albeit sparsely. In the early 90s, Binder began
documenting the facial changes over time in 2 identical
twins living in different countries in what would become
a 19-year case-report.3,4 One twin received BoNTA in
the forehead and glabella regularly every 4 to 6 months
for 19 years, with additional injections in the periorbital
region for the last 8 years of the study. Her sister
received a total of 4 treatment sessions in the forehead,
glabella, and crow’s feet over the length of the entire
study. Neither twin used any topical retinoid prepara-
tion or received any other cosmetic treatments. The
contrast was obvious and striking: the twin treated
regularly displayed no evidence of forehead or glabellar
rhytides at rest, only mild crow’s feet on animation, and
improved skin texture, while the twin treated only
sporadically displayed a greater number and depth of
wrinkles and visible pores (Figure 1). In 2008, Bowler
reported similar results in a case series of 2 individuals
who received 21 to 24 treatment sessions over a 7-year
period every 3 to 6 months for glabellar, forehead, and
periorbital rhytides.5 Both patients exhibited a gradual
reduction in visible rhytides, with eventual effacement
of nonreducible forehead lines.
Dailey studied the impact of repeated treatment cycles on
wrinkle severity and treatment intervals in a 2-year pro-
spective study.6 More than half the participants demon-
strated at least a 1-point improvement on the facial
wrinkle score at maximum contraction at 20 and 26

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 BOTULINUM TOXIN TYPE A IN FACIAL AESTHETICS

Figure 1. A study of identical twins over 19 years demonstrates the treated twin exhibits virtually no forehead rhytides at rest
(A), whereas static rhytides are visible in the sporadically treated twin (B). Similarly, crow’s feet are mild in the treated twin (C),
and deeper in the twin treated sporadically (D). Reproduced with permission from Rivkin A, Binder WJ. Long-term effects of
onabotulinumtoxinA on facial lines: a 19-year experience of identical twins. Dermatol Surg 2015;41(Suppl 1):S64–6.

months. At final treatment, more than 80% of partic-
ipants had at least a 1-point improvement at repose, an
improvement in resting rhytides that was maintained 6
months after treatment by nearly two-thirds of the
patients. Six months after the last treatment, 87% of
patients remained satisfied with the results. These pro-
gressive improvements in wrinkle severity and extended
duration of effect as the number of treatment cycles pro-
gressed suggest that repeated treatments at regular, fixed
intervals may result in continued reduction in the severity
of existing wrinkles and prolonged injection intervals.
The effect of repeated treatment of BoNTA on the
overall appearance of the skin is more difficult to
characterize. In the literature, validated measures for
skin quality focus on specific attributes, such as elas-
ticity and pliability,7,8 hydration,9 or sebum pro-
duction and pore size10–13 after a single treatment with
BoNTA. Some investigators have noted additional
improvements in skin texture and radiance after
multiple, consistent injections with BoNTA. In 1995,
Ascher described a “smoothing” effect—in addition to
wrinkle reduction—in areas treated with BoNTA that
lasted 6 to 9 months after several injections.14 In
Binder and Rivkin’s study, the twin treated regularly
demonstrated improved skin texture on observation
when compared with her sister,4 while Bowler noted
significant improvements in skin quality after multiple

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treatment sessions over 7 years.5 In 2007, Dessy and
colleagues15 reported a statistically significant reduc-
tion in superficial skin texture—including average skin
roughness—after BoNTA in the glabella, and sug-
gested this decrease in roughness likely explained the
observed “smoother and lighter appearance of the
skin after treatment.” This outcome has had an
apparent effect on clinical practice: intradermal
injection of multiple droplets of diluted BoNTA over
a wide area of the face and neck is used increasingly as
a “skin-booster,” producing a reported lifting effect
that has been attributed to improved skin texture and
radiance.16,17
Carruthers assessed the concept of this potential
“smoothing” effect by extracting data from 2 ran-
domized trials and analyzing the response to a single
treatment of BoNTA in 183 patients with mild gla-
bellar lines at rest.18 Nearly 70% of patients transi-
tioned from mild lines at rest to none; 41% still
showed elimination of glabellar lines at repose 120
days after treatment. To determine whether the effects
would persist over repeated treatments, Carruthers
analyzed the results of 3 treatment cycles spaced
4 months apart in 225 patients with mild to severe
glabellar lines at rest.19 Repeated treatment improved
the likelihood of eliminating lines at rest, producing
the smoothing effect that subsequently contributed to
a more youthful appearance and sustained patient
satisfaction.
For the purpose of this paper, we define good skin
quality as the individual, specific skin attributes that
collectively describe skin that appears healthier and
more youthful. We also consider attributes associated
with fibroblasts and dermal extracellular matrix
(ECM) structure, which would include, for example,
skin roughness, laxity and fine lines. In clinical prac-
tice, many of the improvements we have observed are
qualitative and subjective, and refer to a global
impression of youth and vitality, with skin that is
smooth and radiant. This effect may be due, in part, to
a reduction in visible rhytides, a byproduct of regular
BoNTA injections and prolonged muscular chemo-
denervation. Several studies of intradermal injection
of BoNTA have demonstrated a reduction in sebum
production and subsequent improvements in skin
© 2017 by the American Society for Dermatologic Surgery, Inc. Published by Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
HUMPHREY ET AL
oiliness and pore size, possibly by blocking release of
acetylcholine from neuronal cells proximal to seba-
ceous glands.10–13 Acetylcholine stimulates lipogen-
esis in sebocytes by binding to acetylcholine receptors
in a paracrine and autocrine fashion; blocking neu-
ronal acetylcholine release could potentially affect
sebocytes in sebaceous glands. Decreased sweat and
sebaceous gland activity improves the appearance of
the skin.17 However, the observed effect on skin
quality attributes associated with sebocyte and
fibroblast function is suggestive of a potential direct
or indirect effect of BoNTA on these cells types,
perhaps beyond inhibition of acetylcholine release
and muscle relaxation.
Scientific Observation: Neurotoxin, Collagen,
and Biomechanics
The skin maintains its mechanical strength, resilience,
and elasticity due to the presence of collagen and
elastic fibers that are organized and tightly packed
within the ECM, creating a basket-type weave that
runs parallel to the skin surface. Over time, enzymatic
degradation and fragmentation of collagen and elastin
compromise this network; the structural components
within the ECM are no longer interwoven in a func-
tional network but form a disorganized, wide
agglomeration less capable of providing sufficient
support, contributing to skin laxity and wrinkle for-
mation.20 Enzymatic degradation of collagen is greatly
accelerated by extrinsic factors, particularly ultravio-
let (UV) radiation and smoking. Chronic UV exposure
leads to a dramatic loss of collagen and the promotion
of elastosis, with accumulation of abnormal elastin
material, and is a major factor contributing to facial
aging.21,22
Because degradation of collagen has a direct influence
on skin quality and appearance, scientific evidence for
histological changes in collagen structure after treat-
ment with BoNTA is of particular and primary inter-
est. There is some indication that BoNTA injections
stimulate deposition of collagen and lead to a reorga-
nization of the collagen network within the ECM.16,23
In 2008, Chang reported the wrinkle-soothing effects
of intradermal injection of 20 to 25 U of BoNTA in 9
volunteers; histologic examination of skin biopsies
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 BOTULINUM TOXIN TYPE A IN FACIAL AESTHETICS

taken 8 weeks after treatment revealed slight increases
in levels of Procollagen II.16 El-Domyati found both
epidermal and dermal changes after a single injection
of BoNTA in the periorbital region in 16 volunteers.23
Biopsy specimens taken 3 months after treatment
showed collagen fibers oriented in a more organized
and uniform pattern, similar to what is observed in
youthful skin, in contrast to the more disorganized,
fragmented collagen observed in pretreated samples
(Figure 2), while there was no significant overall
increase in the density of collagen Types I and III or
elastin. These histological findings suggest the begin-
nings of a dermal remodeling process in which existing
damaged collagen is replaced with new collagen and
built into more organized, tightly packed collagen
fiber bundles, although it is difficult to know whether
the effect of BoNT on fibroblasts is direct, or if, for
example, relaxation of the underlying muscle stim-
ulates fibroblasts and indirectly induces reorganiza-
tion of the collagen bundles.
Improvements to the structural web of collagen in the
dermis may explain alterations in biomechanical fea-
tures of the skin after treatment with BoNTA.7–9
Bonaparte and Ellis examined elastic recoil and pli-
ability of the skin after only one treatment using low
doses of BoNTA in the glabella, forehead, and lateral
orbit, and found characteristics more in keeping with
younger skin.7,8 Mean pliability significantly increased
from baseline at 2 and 3 months post-treatment across
all sites. Significant improvements in elastic recoil were
noted at 2 months at all sites; by 4 months, only the
glabella maintained a significant increase in elastic
recoil.8 The increases in pliability and elasticity that
returned to baseline measurements by 3 or 4 months
mimics the course of neuromuscular effects of BoNTA.
Similarly, Zhu measured biophysical parameters—
roughness, elasticity, skin hydration, transepidermal
water loss, erythema, and melanin index—in 20 vol-
unteers who received intradermal injection of BoNTA
on one side of the face and noted improvements 3

Figure 2. Dermal collagen and elastin before and after 3 months of BoNTA. Immunoperoxidase staining of skin biopsy
specimens for collagen Types I (A and B) and III (C and D) and elastin (E and F) showing no significant difference in collagen
Types I and III and elastin content between baseline (A, C, and E) and post-treated biopsies (B, D, and F), but with better
organization and more compact collagen fibers after BoNTA injection (B and D). Reproduced with permission from
El-Domyati M, Attia SK, El-Sawy AE, Moftah NH, et al. The use of botulinum toxin-a injection for facial wrinkles:
a histological and immunohistochemical evaluation. J Cosmet Dermatol 2015;14:140–4.

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months after treatment in all parameters except ery-
thema and melanin compared with control.9 Of par-
ticular interest is the apparent spike in effect between
4 weeks—when the observed parameters between
treatment and control groups were about the same
magnitude—and 3 months, which appears to align
with the timeline of improvements noted by Bona-
parte7,8 and the clinical duration of effect on rhytides.
Many factors may be at play. It has been suggested that
percutaneous injury with needles initiates the wound-
healing cascade, stimulating neocollagenesis.24,25 It is
possible that relaxation of the underlying muscle
stimulates reorganization of the collagen network.
Fibroblasts are known to respond to mechanical ten-
sion.26 Chemical denervation of the skin could also
inhibit neurogenic inflammation and prevent kerati-
nocyte and fibroblast activation.27,28 More interesting,
however, is the possibility that injection of BoNTA has
a direct influence on fibroblasts themselves.
Botulinum Toxin and Potential
Fibroblastic Response
As skin ages, the number of dermal fibroblasts
decreases, while matrix metalloproteinases (MMPs)—
catalysts for collagen degradation—increase.29
Remodeling of the ECM requires direct activation of
fibroblasts, and this is the question at hand: can this be
achieved via BoNTA? Oh studied in vitro fibroblast
proliferation, collagen production, and expression of
certain MMPs after application of small doses of
BoNTA on human dermal fibroblasts.29 Cultured
fibroblasts demonstrated increased production of
procollagen Type I carboxy-terminal peptide, which
indirectly reflects overall Collagen I levels, and signif-
icantly decreased expression of 2 MMPs. Levels of 2
proalpha collagen chains—alpha-1, the major com-
ponent of Type I collagen, and alpha-2—were also
higher after treatment, suggesting that BoNTA has the
potential to promote dermal remodeling.
Permatasari30 demonstrated similar findings in pho-
toaged dermal fibroblasts. Natural enzymatic degra-
dation of collagen is greatly accelerated by extrinsic
factors, particularly UV radiation.31 The authors
incubated ultraviolet B (UVB)-stressed fibroblasts with
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HUMPHREY ET AL
BoNTA at 3 different concentrations, and evaluated the
effects on collagen production, degradation, and
senescence-related markers to investigate the
antiphotoaging potential of BoNTA. In UVB-irradiated
dermal fibroblasts, BoNTA increased the expression of
Collagen I and III and decreased secretion of 2 MMPs.
Taken together, these results suggest that BoNTA has
the potential to promote remodeling of photoaged skin
by directly stimulating collagen production and
decreasing enzymes that have the ability to degrade
nearly all ECM components, including collagen.30
Research in wound healing and scar prevention yields
a growing body of evidence that BoNTA reduces
abnormal fibroblast proliferation and apoptosis,
which drives the formation of hypertrophic scars and
keloids.32–36 BoNTA has long been used in surgical
settings to intentionally reduce tension around the
edges of a wound with the goal of minimizing the
resulting scar.37–40 It has been postulated that this
success as surgical adjunct is due to tension-relieving
properties together with direct inhibitory effects on
fibroblasts and transforming growth factor (TGF)-b1
expression.33,40 TGF-b1 is considered the main regu-
lator in the pathogenesis of hypertrophic scars and is
associated with excessive deposition of scar tissue and
fibrosis.32,34,36 Cellular analysis of hypertrophic scar-
ring successfully treated with BoNTA reveals reduced
fibroblast proliferation and expression of TGF-b1.
Direct inhibition of fibroblast differentiation occurred
in hypertrophic—but not normal—tissue samples,36
suggesting that BoNTA may correct the imbalance in
cellular dynamics caused by overabundance of pro-
liferation and lack of cellular apoptosis.33
Like hypertrophic scars, keloids result from over-
zealous proliferation of fibroblasts in response to
a wound. The growth of keloid fibroblasts is rapid and
invasive, spreading beyond the borders of the initial
site of injury.35 Attempts to use BoNTA for the treat-
ment of established keloids has yielded conflicting
results.41–43 Zhibo injected lesions at 3-month inter-
vals for up to 9 months in 12 patients and observed fair
to excellent results in scar regression at 1-year follow-
up.41 The authors proposed a reduction of TGF-b1
expression and decreased fibroblast proliferation as
the underlying mechanism of action. However,
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 BOTULINUM TOXIN TYPE A IN FACIAL AESTHETICS
Gauglitz42,43 observed no scar reduction or in vitro
effects on TGF-b expression or fibroblasts after
treatment with BoNTA. Xiaoxue examined changes in
gene expression in an attempt to unravel the possible
mechanism of action of BoNTA inhibition of invasive
growth of keloid-causing fibroblasts.35 Analyses from
microarray and quantitative real-time polymerase
chain reaction showed modulation of expression of 5
genes relevant to cell regulation and invasive growth of
fibroblasts—including the downregulation of MMP-1
and TGF-b1—in response to BoNTA exposure.
Hypertrophic scars and keloids both represent
a derailment of the protective wound-healing process.
Research examining fibroblastic response to BoNTA
suggests the neurotoxin may participate in the regu-
lation of some genes responsible for proper cell func-
tioning, restoring order to a disordered and fractured
matrix, and it is this careful balance in cellular
dynamics that produces visible improvements in the
skin that become more pronounced after repeated
treatment.
Conclusions
Clinical observation of progressive wrinkle reduction
and qualitative improvements in a number of skin
quality attributes that accumulate with more frequent
injections of BoNTA suggest an ongoing process of
repair leading to prolonged and cumulative effects.
Repeated injections of BoNTA appears to reduce the
formation of wrinkles with an effect that also appears
to be sustained over time and enhanced with regular
treatments. Resting rhytides improve, and clinicians
have noted that regularly treated skin takes on
a smoother, more radiant appearance. Bio-
mechanically, skin is more pliable and elastic. Even
a single treatment has been shown to improve physi-
ological properties of the skin associated with youth
that are difficult to explain by muscle relaxation alone.
Histological investigations provide evidence of direct
effects on fibroblasts and connective tissue. Injection
of BoNTA appears to stimulate collagen production,
decrease enzymatic degradation of ECM components,
and regulate cell apoptosis and fibroblast proliferation
in fibrotic tissue. Although there are likely other con-
tributing mechanisms that have not been elucidated,
S250 DERMATOLOGIC SURGERY
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the literature paints a circumstantial picture framed by
physiologic evidence that regular, consistent use of
BoNTA provides sustained and cumulative improve-
ments in the quality and appearance of the skin.
The implications could have a profound effect on
clinical practice. Future research might focus on
characterizing those skin qualities that sparked the
original hypothesis and defining what constitutes
“regular treatment” in order to study these apparent
benefits and confirm the validity of our anecdotal
clinical observations.
References
1. Carruthers JD, Carruthers JA. Treatment of glabellar frown lines with
C. botulinum-A exotoxin. J Dermatol Surg Oncol 1992;18:17–21.
2. American Society of Plastic Surgeons (2017). 2016 Plastic Surgery
Statistics Report. Available from: www.plasticsurgery.
org/news/plastic-surgery-statistics. Accessed June 6, 2017.
3. Binder WJ. Long-term effects of botulinum toxin type A (Botox) on
facial lines: a comparison in identical twins. Arch Facial Plast Surg
2006;8:426–31.
4. Rivkin A, Binder WJ. Long-term effects of onabotulinumtoxinA on
facial lines: a 19-year experience of identical twins. Dermatol Surg
2015;41(Suppl 1):S64–6.
5. Bowler PJ. Dermal and epidermal remodeling using botulinum toxin
type A for facial, non reducible, hyperkinetic lines: two case studies. J
Cosmet Dermatol 2008;7:241–4.
6. Dailey RA, Philip A, Tardie G. Long-term treatment of glabellar
rhytides using onabotulinumtoxina. Dermatol Surg 2011;37:918–28.
7. Bonaparte JP, Ellis D. Skin biomechanical changes after injection of
onabotulinum toxin A: prospective assessment of elasticity and
pliability. Otolaryngol Head Neck Surg 2014;150:949–55.
8. Bonaparte JP, Ellis D. Alterations in the elasticity, pliability, and
viscoelastic properties of facial skin after injection of onabotulinum
toxin A. JAMA Facial Plast Surg 2015;17:256–63.
9. Zhu J, Ji X, Xu Y, Liu J, et al. The efficacy of intradermal injection of
type A botulinum toxin for facial rejuvenation. Dermatol Ther 2017;30:
e12433.
10. Shah AR. Use of intradermal botulinum toxin to reduce sebum
production and facial pore size. J Drugs Dermatol 2008;7:847–50.
11. Li ZJ, Park SB, Sohn KC, Lee Y, et al. Regulation of lipid production by
acetylcholine signalling in human sebaceous glands. J Dermatol Sci
2013;72:116–22.
12. Rose AE, Goldberg DJ. Safety and efficacy of intradermal injection of
botulinum toxin for the treatment of oily skin. Dermatol Surg 2013;39:
443–8.
13. Min P, Xi W, Grassetti L, Trisliana Perdanasari A, et al. Sebum
production alteration after botulinum toxin type A injections for the
treatment of forehead rhytides: a prospective randomized double-blind
dose-comparative clinical investigation. Aesthet Surg J 2015;35:600–10.
14. Ascher B, Klap P, Marion MH, Chanteloub F. Botulinum toxin in the
treatment of frontoglabellar and periorbital wrinkles. An initial study
[in French]. Ann Chir Plast Esthet 1995;40:67–76.

15. Dessy LA, Mazzocchi M, Rubino C, Mazzarello V, et al. An objective
assessment of botulinum toxin A effect on superficial skin texture. Ann
Plast Surg 2007;58:469–73.
16. Chang SP, Tsai HH, Chen WY, Lee WR, et al. The wrinkles soothing
effect on the middle and lower face by intradermal injection of
botulinum toxin type A. Int J Dermatol 2008;47:1287–94.
17. Wu WT. Microbotox of the lower face and neck: evolution of
a personal technique and its clinical effects. Plast Reconstr Surg 2015;
136(5 Suppl):92S–100S.
18. Carruthers A, Carruthers J, Lei X, Pogoda JM, et al.
OnabotulinumtoxinA treatment of mild glabellar lines in repose.
Dermatol Surg 2010;36(Suppl 4):2168–71.
19. Carruthers A, Carruthers J, Fagien S, Lei X, et al. Repeated
onabotulinumtoxinA treatment of glabellar lines at rest over three
treatment cycles. Dermatol Surg 2016;42:1094–101.
20. Landau M, Fagien S. Science of hyaluronic acid beyond filling:
fibroblasts and their response to the extracellular matrix. Plast Reconstr
Surg 2015;136(5 Suppl):188S–195S.
21. Baumann L. Skin ageing and its treatment. J Pathol 2007;211:241–51.
22. Hughes MC, Williams GM, Baker P, Green AC. Sunscreen and
prevention of skin aging: a randomized trial. Ann Intern Med 2013;
158:781–90.
23. El-Domyati M, Attia SK, El-Sawy AE, Moftah NH, et al. The use of
botulinum toxin-a injection for facial wrinkles: a histological and
immunohistochemical evaluation. J Cosmet Dermatol 2015;14:140–4.
24. Fernandes D, Signorini M. Combating photoaging with percutaneous
collagen induction. Clin Dermatol 2008;26:192–9.
25. Kapoor RL, Shome D, Jain V, Dikshit R. Facial rejuvenation after
intradermal botulinum toxin: is it really the botulinum toxin or is it the
pricks? Dermatol Surg 2010;36(Suppl 4):2098–105.
26. Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, et al. Myofibroblasts
and mechano-regulation of connective tissue remodelling. Nat Rev Mol
Cell Biol 2002;3:349–63.
27. Werner S, Krieg T, Smola H. Keratinocyte-fibroblast interactions in
wound healing. J Invest Dermatol 2007;127:998–1008.
28. Oh HM, Chung ME. Botulinum toxin for neuropathic pain: a review of
the literature. Toxins (Basel) 2015;7:3127–54.
29. Oh SH, Lee Y, Seo YJ, Lee JH, et al. The potential effect of botulinum
toxin type A on human dermal fibroblasts: an in vitro study. Dermatol
Surg 2012;38:1689–94.
30. Permatasari F, Hu YY, Zhang JA, Zhou BR, et al. Anti-photoaging
potential of botulinum toxin type A in UVB-induced premature
senescence of human dermal fibroblasts in vitro through decreasing
senescence-related proteins. J Photochem Photobiol B 2014;133:115–23.
31. Quan T, Little E, Quan H, et al. Elevated matrix metalloproteinases and
collagen fragmentation in photodamaged human skin: impact of altered
© 2017 by the American Society for Dermatologic Surgery, Inc. Published by Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
HUMPHREY ET AL
extracellular matrix microenvironment on dermal fibroblast function. J
Invest Dermatol 2013;133:1362–6.
32. Zhibo X, Miaobo Z. Botulinum toxin type A affects cell cycle
distribution of fibroblasts derived from hypertrophic scar. J Plast
Reconstr Aesthet Surg 2008;61:1128–9.
33. Xiao Z, Zhang F, Cui Z. Treatment of hypertrophic scars with
intralesional botulinum toxin type A injections: a preliminary report.
Aesthet Plast Surg 2009;33:409–12.
34. Xiao Z, Zhang M, Liu Y, Ren L. Botulinum toxin type
a inhibits connective tissue growth factor expression in
fibroblasts derived from hypertrophic scar. Aesthet Plast Surg 2011;
35:802–7.
35. Xiaoxue W, Xi C, Zhibo X. Effects of botulinum toxin type A on
expression of genes in keloid fibroblasts. Aesthet Surg J 2014;34:154–9.
36. Jeong HS, Lee BH, Sung HM, Park SY, et al. Effect of botulinum toxin
type A on differentiation of fibroblasts derived from scar tissue. Plast
Reconstr Surg 2015;136:171e–8e.
37. Sherris DA, Gassner HG. Botulinum toxin to minimize facial scarring.
Facial Plast Surg 2002;18:35–9.
38. Gassner HG, Sherris DA. Chemoimmobilization: improving
predictability in the treatment of facial scars. Plast Reconstr Surg 2003;
112:1464–6.
39. Ziade M, Domergue S, Batifol D, Jreige R, et al. Use of botulinum toxin
type A to improve treatment of facial wounds: a prospective
randomised study. J Plast Reconstr Aesthet Surg 2013;66:209–14.
40. Kim YS, Lee HJ, Cho SH, Lee JD, et al. Early postoperative treatment
of thyroidectomy scars using botulinum toxin: a split-scar, double-
blind randomized controlled trial. Wound Repair Regen 2014;22:
605–12.
41. Zhibo X, Miaobo Z. Intralesional botulinum toxin type A injection as
a new treatment measure for keloids. Plast Reconstr Surg 2009;124:
275e–7e.
42. Gauglitz GG, Bureik D, Dombrowski Y, Pavicic T, et al. Botulinum
toxin A for the treatment of keloids. Skin Pharmacol Physiol 2012;25:
313–8.
43. Gauglitz GG. Management of keloids and hypertrophic scars:
current and emerging options. Clin Cosmet Investig Dermatol
2013;6:103–14.
Address correspondence and reprint requests to: Shannon
Humphrey, MD, Carruthers & Humphrey Cosmetic
Dermatology, University of British Columbia, 943 W
Broadway #820, Vancouver, BC V5Z 4E1, Canada, or
e-mail: shannon@carruthers-humphrey.com
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