Mesotherapy with an Intradermal Hyaluronic Acid Formulation

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ORIGINAL ARTICLE NON-SURGICAL AESTHETIC
Mesotherapy with an Intradermal Hyaluronic Acid Formulation
for Skin Rejuvenation: An Intrapatient, Placebo-Controlled,
Long-Term Trial Using High-Frequency Ultrasound
Aurora Tedeschi • Francesco Lacarrubba •
Giuseppe Micali
Received: 11 August 2014 / Accepted: 16 November 2014 / Published online: 25 December 2014
� Springer Science+Business Media New York and International Society of Aesthetic Plastic Surgery 2014
Abstract Mesotherapy with hyaluronic acid (HA) is a
treatment approach currently used for skin rejuvenation.
High-frequency ultrasound (20–100 MHz) is a non-inva-
sive technique that has been used to evaluate age-related
dermal changes. The presence and the degree of a typical
subepidermal low-echogenic band (SLEB) are photoaging
related: the lower the SLEB echogenicity, the higher the
photoaging. The aim of this trial was to evaluate, through
ultrasound imaging, the long-term effects of microinjec-
tions of HA on SLEB echogenicity. Twenty-two women
with clinical and ultrasound signs of moderate photoaging
were enrolled in the study. Treatment consisted of multiple
microinjections of HA salts of biotechnological origin on
the dorsum of one hand, once weekly for 4 weeks and,
successively, once monthly for 4 months (group A) or
9 months (group B). The dorsum of the other hand of each
subject was injected with saline solution and used as a
control. In all subjects, high-frequency ultrasound
(22 MHz) was performed to evaluate SLEB echogenicity
changes during treatment. Eighteen out of 22 patients
completed the study. At the end of 4 weeks, an ultrasound
increase of dermal echogenicity was observed in 13 sub-
jects (seven of group A and six of group B), which we
considered as ‘‘responders’’. In these patients, the Student’s
t-test showed a significant increase from baseline of SLEB
pixel numbers of ?24 % (P \ 0.01) versus ?6 % with
placebo. In the same subjects, after an additional 4 months
of monthly injections, the mean increase was ?18 %
(P \ 0.05) versus ?4 % with placebo. In patients from
group B that completed 10 months of treatment, the
increase from baseline of SLEB pixel numbers was ?18 %
(P \ 0.05) versus 0 % with placebo. Our study suggests
that mesotherapy with HA may effectively improve skin
aging and photoaging, as supported by quantifiable ultra-
sound data showing significant changes in SLEB density
over time.
Level of Evidence II This journal requires that authors
assign a level of evidence to each article. For a full
description of these Evidence-Based Medicine ratings,
please refer to the Table of Contents or the online
Instructions to Authors www.springer.com/00266.
Keywords Mesotherapy � Hyaluronic acid � Ultrasound �
Skin rejuvenation
Introduction
Hyaluronic acid (HA) is an essential component of the
extracellular matrix, influencing skin water content and,
consequently, skin flexibility, hydration, and firmness.
Chemically, HA is a glycosaminoglycan, derived from
polymerization of the monosaccharide d-glucuronic acid
combined with n-acetyl-glucosamine [1, 2]. HA promotes
fibroblast proliferation and migration as well as collagen
synthesis. Decreased HA correlates with skin aging and
photoaging [3].
Skin aging is related to intrinsic (genetically-driven,
physiological changes over time) and extrinsic factors,
such as chronic ultraviolet (UV) light exposure (respon-
sable of photoaging), pollutants, and smoking. Alterations
of connective tissue components, such as reduction of
fibroblasts, collagen, and elastic fibers, result in visible skin
morphologic changes, with onset of wrinkling, sagging,
loss of elasticity, and xerosis [3]. In the past years,
A. Tedeschi � F. Lacarrubba � G. Micali (&)
Dermatology Clinic, University of Catania, A.O.U. Policlinico-
Vittorio Emanuele, Via Santa Sofia, 78, 95123 Catania, Italy
e-mail: cldermct@nti.it
123
Aesth Plast Surg (2015) 39:129–133
DOI 10.1007/s00266-014-0432-1
http://www.springer.com/00266
‘‘dermatoporosis’’ has been proposed as a term for age-
related skin changes, especially those related to decreased
HA content along with a decreased expression of major cell
surface hyaluronate receptors for CD44 [4]. The use of
intradermal HA injections represents one of the most
popular treatments currently used in cosmetic dermatology
for skin rejuvenation, although studies supporting its effi-
cacy besides clinical evidence are limited [5, 6].
High-frequency ultrasound (20–100 MHz) is a non-
invasive technique [7, 8] that has been used to evaluate
age-related skin changes. In particular, a subepidermal
low-echogenic band (SLEB), located immediately below the
epidermal entrance echo, has been identified as an indicator
of photoaged skin [9, 10]. Moreover, it has been demon-
strated that SLEB echogenicity inversely correlates with
chronic UV radiation exposure: the lower the SLEB ech-
ogenicity, the greater the photoaging. Consequently, ultra-
sound has been used to evaluate efficacy of anti-aging
products [10–13]. In a previous study, we used ultrasound to
evaluate the treatment of multiple intradermal injections of
HA as a mesotherapy agent in a four-week trial [14]. In
consideration of the promising results obtained from this
study, we decided to perform a long-term evaluation.
Materials and Methods
Twenty-two women (range 36–65 years, mean age 50.5)
with clinical and ultrasound signs of moderate photoaging
were enrolled in this intrapatient placebo-controlled trial.
The protocol was approved by our local institutional
review board. Inclusion criteria were as follows: female
gender, age ranging from 35 to 65 years, and presence of
physical signs of moderate photoaging on the dorsum of
the hands, with evidence of SLEB at ultrasound evaluation.
Exclusion criteria were superficial chemical peels per-
formed within 4 weeks, history of allergic and/or irritant
contact hand dermatitis, systemic diseases, psychiatric ill-
ness, and pregnancy. Informed consent from all partici-
pants was obtained. Each subject was treated with multiple
intradermal microinjections (Fig. 1) of 1 ml HA salts
(sodium chloride, chloride sodium phosphate) of biotech-
nological origin with a molecular weight of 1.000 kDa
(Viscoderm�) at a concentration of 20 mg/ml to the dor-
sum of left hand, and with the same amount of saline
solution (placebo) to the dorsum of the right hand. Par-
ticipants were randomly assigned to one of two groups:
group A received 4 weekly injections followed by 4
monthly injections (end point: 6 months; 5 months treat-
ments, 1-month follow up); group B received 4 weekly
injections followed by 9 monthly injections (end point:
11 months; 10 months treatments, 1-month follow up)
(Fig. 1). Each treatment consisted of a maximum total
injected volume of 1 ml (approximately 30 injections) of
HA or saline solution in both left and right hands,
respectively.
Fig. 1 Intradermal microinjections of HA salts to the dorsum of left
hand
Fig. 2 Treatment schedule:
microinjections of HA salts of
biotechnological origin (total
amount injected in each session:
20 mg/ml) on the dorsum of one
hand once weekly for 4 weeks
and, successively, once monthly
for 4 months (for a total of
5 months of treatment, group A)
or 9 months (for a total of
10 months of treatment, group
B)
130 Aesth Plast Surg (2015) 39:129–133
123
In all subjects, ultrasound evaluation was performed at
each visit by the same investigator (FL), under constant
environmental conditions, on a target area corresponding to
the second metacarpal web space of the hands, immedi-
ately before and one week after each treatment, to evaluate
SLEB echogenicity changes throughout the study duration.
Ultrasound was avoided immediately after HA adminis-
tration, because of temporary microwheal formation after
microinjection. Cross-sectional B-mode scans were
obtained by a 22-MHz ultrasound system. The gain setting
was keptat a constant level (40 dB). For each examined
field, the amplitudes of echoes of single image elements
(pixels) of the SLEB were ascribed to a numerical scale
(0–255) and mean gray values were quantified with ImageJ
public domain software. The treatment schedule and
ultrasound evaluations are summarized in Fig. 2. Student
t-test was used for statistical analysis.
Results
Eighteen out of 22 patients completed the study. At the end
of 4 weeks, an ultrasound increase of dermal echogenicity
was observed in 13 subjects (seven of group A and six of
group B), which we considered as ‘‘responders.’’ In these
patients, a significant increase from baseline of SLEB pixel
numbers corresponding to ?24 % (P \ 0.01) versus ?6 %
Table 1 Results from group A
Group
A
Treatment SLEB
echogenicity
at baseline
(pixels/area)
SLEB
echogenicity
after 4 weeks
(pixels/area)
SLEB
echogenicity
after 6 months
(pixels/area)
Pt. 1 HA 9.58 14.49 10.35
Placebo 9.15 12.73 9.92
Pt. 3 HA 10.81 12.2 10.79
Placebo 10.84 11.88 10.71
Pt. 6 HA 11.33 12.89 11.22
Placebo 14.05 13.38 12.55
Pt. 7 HA 12.38 12.55 12.44
Placebo 11.38 13.82 11.34
Pt. 9 Drop out
Pt. 10 HA 12.29 13.45 14.72
Placebo 13.57 14.1 13.12
Pt. 11 HA 12.29 13.77 17.12
Placebo 11.74 13.96 17.34
Pt. 14 HA 8.65 13.81 10.27
Placebo 9.74 9.79 9.1
Pt. 16 HA 13.51 11.76 13.89
Placebo 11.78 11.54 11.31
Pt. 18 HA 12.68 13.03 12.98
Placebo 10.78 10.56 11.34
Pt. 19 HA 11.81 13.51 12.03
Placebo 11.13 12.89 11.2
Table 2 Results from group B
Group B Treatment SLEB echogenicity
at baseline
(pixels/area)
SLEB echogenicity
after 4 weeks
(pixels/area)
SLEB echogenicity
after 6 months
(pixels/area)
SLEB echogenicity
after 11 months
(pixels/area)
Pt. 2 HA 9.32 10.74 15.55 11.59
Placebo 11.19 10.94 13.11 10.91
Pt. 4 Drop out
Pt. 5 Drop out
Pt. 8 HA 9.27 12.83 13.02 11.52
Placebo 14.79 13.34 16.01 13.02
Pt. 12 HA 9.03 11.04 9.81 11.76
Placebo 12.83 11.51 11.23 11.82
Pt. 13 HA 11.28 13.24 12.71 12.77
Placebo 11.78 13.85 13.25 12.88
Pt. 15 HA 11.2 12.81 12.11 12.54
Placebo 11.18 10.83 11.98 12.43
Pt. 17 Drop out
Pt. 20 HA 8.02 12.43 9.63 8.68
Placebo 10.91 13.81 10.66 10.76
Pt. 21 HA 12.31 12.38 11.87 11.65
Placebo 14.5 14.63 12.36 14.19
Pt. 22 HA 10.23 10.8 10.32 12.25
Placebo 11.82 11.3 9.77 11.89
Aesth Plast Surg (2015) 39:129–133 131
123
with placebo was observed. In the same subjects, after an
additional 4 months of monthly injections, the mean
increase was ?18 % (P \ 0.05) versus ?4 % with pla-
cebo. In patients of group B, that completed 10 months of
treatment, the mean increase of pixel numbers of the SLEB
from baseline was ?18 % (P \ 0.05) versus 0 % with
placebo. Ultrasound results are summarized in Tables 1
and 2. No complications resulted from the treatments, but a
few, small ecchymoses occurred in some cases that
improved after 4–5 days.
Discussion
HA, used both as mesotherapy and/or filler, has become
increasingly popular in the management of skin aging and
photoaging. Mesotherapy is a well-established esthetic
technique, introduced in France in 1952 [15]. It is
reportedly useful in the management of a variety of con-
ditions mainly in rheumatology and aesthetic medicine [5].
Mesotherapy consists of subcutaneous injection of a mix-
ture of biocompatible compounds (including nutrients,
hormones, vitamins, and enzymes) that are delivered in
small doses, by means of very fine needles, directly over/
near the target areas [15]. This technique allows the intro-
duction of ingredients that are released over a prolonged
period of time to the surrounding tissues through a depot-
like effect [16, 17]. Mesotherapy in skin rejuvenation is
intended to maintain and/or restore skin texture [18, 19].
Some clinical studies have demonstrated that intradermal
HA administration increases tissue water retention as well
as fibroblast activity [20, 21]. In particular, it has shown to
stimulate fibroblasts to express collagen type 1 (Col-1),
matrix metalloprotease-1 (MMP-1), tissue inhibitor of
Fig. 3 Subject 14 (group A): increase of SLEB echogenicity was
observed after the first 4 weeks of treatment with HA as well as after
6 months (see changes in areas limited by yellow lines)
Fig. 4 Subject 15 (group B): ultrasound increase of SLEB echoge-
nicity was observed after the first 4 weeks of treatment with HA as
well as after 11 months (see changes in areas limited by yellow lines)
132 Aesth Plast Surg (2015) 39:129–133
123
matrix metalloproteinase-1 (TIMP) [22–23]. A study of 50
subjects supported the safety and efficacy of HA meso-
therapy in the prevention and treatment of skin aging, and
showed a decrease of IL1b, IL6, MMP1 as well as an
increase in Col-1 [23].
Our study confirms that mesotherapy with HA may be
useful in the treatment of skin aging and photoaging, as
supported by quantifiable ultrasound data showing signifi-
cant changes in SLEB density over time (Figs. 3, 4). After
4 weeks (1 injection/week), in 13 out of 18 patients from
both group A and B, a statistically significant improvement
of the SLEB echogenicity (mean ?24 %) was observed in
the HA-treated hand, compared with slight improvement
(mean ?6 %) in the contralateral hand treated with normal
saline solution. In the same 13 patients, after 4 additional
months of treatment with monthly injections, the
improvement from baseline was ?18 % (HA-treated hand)
versus ?4 % (contralateral hand). Interestingly, in patients
of group B that completed 10 months of treatment, the
improvement in the HA-treated hand remained steady
(mean ?18 %), in contrast to a progressive reset (0 %),
observed in the contralateral hand. These data confirm an
improvement of SLEB echogenicity over time determined
by HA injections. Considering the minimal SLEB change
from saline solution injections, a placebo effect as well as a
hypothetic role of trauma is ruled out.
In conclusion, the cause of increased echogenicity of
SLEB resulting from multiple mesotherapy injections with
HA is unknown. It may likely be due to several factors
including increased density or rearrangement of dermal
collagen fibers after fibroblast activation or a non-specific
inflammatory reaction. Histopathology investigations will
definitively clarify this issue.
Acknowledgments The authors wish to thank Dr. Guido Robecchi
for his invaluable assistance.
Conflict of interest The authors declare that they have no conflicts
of interest to disclose.
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	Mesotherapy with an Intradermal Hyaluronic Acid Formulation for Skin Rejuvenation: An Intrapatient, Placebo-Controlled, Long-Term Trial Using High-Frequency Ultrasound
	Abstract
	Level of Evidence II
	Introduction
	Materials and Methods
	Results
	Discussion
	Acknowledgments
	References