Efficacy of mesotherapy in facial rejuvenation: A histological and immunohistochemical evaluation

Abstract

Mesotherapy, commonly known as "biorejuvenation" or "biorevitalization", is a technique used to rejuvenate the skin by means of a transdermal injection of a multivitamin solution and natural plant extracts that are thought to improve the signs of skin aging.
This prospective study aimed to evaluate the clinical effect of mesotherapy applied to periorbital wrinkles and to quantitatively evaluate histological changes in the skin occurring in response to the same treatment.
Six volunteers with Fitzpatrick skin types III or IV and Glogau class I-III wrinkles were subjected to a three-month course of mesotherapy injections in the periocular area (six sessions administered at two-week intervals). Standard photographs and skin biopsies were obtained from the treatment area at baseline, at the end of treatment, and at three months post-treatment. Quantitative evaluation of collagen types I, III, and VII, newly synthesized collagen, total elastin, and tropoelastin was performed using a computerized morphometric analysis.
The clinical evaluation of volunteers at baseline, end of treatment, and three months post-treatment revealed no significant differences. Histological and immunostaining analysis of collagen types I, III, and VII, newly synthesized collagen, total elastin, and tropoelastin showed no statistically significant changes (P > 0.05) after mesotherapy injection.
The present study indicates that mesotherapy for skin rejuvenation does not result in statistically significant histological changes or clinical improvement.

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Efficacy of mesotherapy in facial rejuvenation: a histological
and immunohistochemical evaluation
Moetaz El-Domyati
1
,MD, Tarek S. El-Ammawi
1
,MD, Osama Moawad
2
,MD,
Hasan El-Fakahany
1
,MD, Walid Medhat
1,3
,MD,M
~
yG. Mahoney
3
,PhD, and
Jouni Uitto
3
,MD, PhD
1
Department of Dermatology, Al-Minya
University, Al-Minya, Egypt,
2
Moawad
Skin Institute for Laser, Cairo, Egypt,
and
3
Department of Dermatology and
Cutaneous Biology, Thomas Jefferson
University, Philadelphia, PA, USA
Correspondence
Moetaz El-Domyati, MD
Department of Dermatology
STDs and Andrology
Al-Minya University
2 Obour Buildings
Salah Salem Street Apt 53
Nasr City
Cairo
Egypt
E-mails: m_domyati@hotmail.com,
moetazeldomyati@yahoo.com,
moetazeldomyati@gmail.com
Funding: This work was supported by a
grant from the Cultural and Educational
Bureau of Egypt to WM, and a National
Institutes of Health grant to JU (R01
AR28450).
Conflicts of interest: None.
Abstract
Background Mesotherapy, commonly known as ‘‘biorejuvenation’’ or ‘‘biorevitalization’’, is
a technique used to rejuvenate the skin by means of a transdermal injection of a multivita-
min solution and natural plant extracts that are thought to improve the signs of skin aging.
Objectives This prospective study aimed to evaluate the clinical effect of mesotherapy
applied to periorbital wrinkles and to quantitatively evaluate histological changes in the skin
occurring in response to the same treatment.
Methods Six volunteers with Fitzpatrick skin types III or IV and Glogau class I–III wrinkles
were subjected to a three-month course of mesotherapy injections in the periocular area
(six sessions administered at two-week intervals). Standard photographs and skin biopsies
were obtained from the treatment area at baseline, at the end of treatment, and at three
months post-treatment. Quantitative evaluation of collagen types I, III, and VII, newly syn-
thesized collagen, total elastin, and tropoelastin was performed using a computerized mor-
phometric analysis.
Results The clinical evaluation of volunteers at baseline, end of treatment, and three
months post-treatment revealed no significant differences. Histological and immunostaining
analysis of collagen types I, III, and VII, newly synthesized collagen, total elastin, and
tropoelastin showed no statistically significant changes (P> 0.05) after mesotherapy
injection.
Conclusions The present study indicates that mesotherapy for skin rejuvenation does not
result in statistically significant histological changes or clinical improvement.
Introduction
Aging is a complex, multifactorial process that affects
every organ in the body, including the skin.
1,2
Clinically,
aged skin is characterized as thin, dry, and pale, with
noticeable wrinkles and decreased elasticity.
3,4
The histo-
logical changes associated with aging in skin include the
accumulation of elastotic material in the papillary and
mid-dermis, a process known as solar elastosis, and quan-
titative changes in collagen, which are reflected in a
decline in biosynthesis and content.
4
Mesotherapy, initially described in France by Michel
Pistor,
5
is one of the modalities recently used to rejuve-
nate and tone the skin.
6,7
It involves the non-invasive
transdermal injection of vitamins, enzymes, hormones,
hyaluronic acid, and natural plant extracts into the skin
to stimulate the biosynthetic ability of fibroblasts and
facilitate interaction between cells and is intended to
increase collagen and elastin production.
8,9
Different
injection techniques can be used in mesotherapy: (i) the
intra-epidermal technique; (ii) the papular technique, in
which reagents are injected into the dermo–epidermal
junction; (ii) the nappage method, in which injections
penetrate to a depth of 2–4 mm and are delivered at an
angle of 30–60; and (iv) point-by-point injection into the
deep dermis.
10,11
Over the past few years, the use of mesotherapy for
skin rejuvenation has increased rapidly at a relatively high 913
ª2012 The International Society of Dermatology International Journal of Dermatology 2012, 51, 913–919

financial cost to patients. As no in-depth, evidence-based
studies have explored the safety or efficacy of mesother-
apy, questions remain about the scientific validity of this
popular approach. The purpose of this study was to eval-
uate the clinical effects of this treatment and to objec-
tively quantify the corresponding histological changes
associated with mesotherapy injection as a non-invasive
method of skin rejuvenation.
Materials and methods
Volunteers and treatment protocol
Six female volunteers with Fitzpatrick skin types III or IV and
Glogau class I–III wrinkles
12
were subjected to three months of
treatment with mesotherapy injection (delivered in six sessions
held at 2-week intervals). The volunteers were recruited from
the dermatology outpatient clinic at Al-Minya University
Hospital, Al-Minya, Egypt, which they had attended for
treatment of periorbital wrinkles. Their mean age was
43.1 ± 4.7 years (range: 37–49 years). The treatment and study
details were fully explained to the subjects, all of whom signed
informed consent forms pertaining to treatment and participation
in the study, which included consent to photography and skin
biopsy before treatment, at three months (end of treatment),
and at six months after the start of treatment (three months
post-treatment).
Mesotherapy injection is a minimally painful procedure and
requires no anesthesia. Before treatment, the volunteer was
positioned for treatment and the periorbital area on both sides
of the face (treatment area) was marked. The sterile solutions
used for injection were composed of a cocktail of a multivitamin
solution and non-cross-linked, high-viscosity hyaluronic acid,
provided in two separate vials in a packaged kit (Revitacare

,
Bio-Revitalisation; Laboratoire Revitacare, Saint Ouen
l’Aumo
ˆne, France). The first vial contained 4 ml of non-cross-
linked hyaluronic acid at 1% biotechnological origin and water
for injectable preparation. The second vial contained 10 ml of a
multivitamin solution of retinol 10,000 IU (5.5 mg), thiamine
4 mg, riboflavin 0.6 mg, nicotinamide 20 mg, dexpanthenol
8 mg, pyridoxine 4 mg, ascorbic acid 100 mg, ergocalciferol
2000 IU (2 mg), tocopherol 4 mg, Solutol

HS 15.52 mg, and
water for injectable preparation. Materials were stored in a
refrigerator, mixed immediately before injection, and transferred
to 1-ml syringes to accommodate the high viscosity of the
hyaluronic acid. For each volunteer, the injection material was
prepared and mixed as a diluted suspension of multivitamin
solution for injection and non-cross-linked, high-viscosity
hyaluronic acid at a ratio of 9 : 1.
The injection process was performed using a meso-system
gun [Anti-Aging Medical Systems (AAMS), Montrodat, France]
controlled by a microprocessor with eight different injection
programs. The gun maintained automatic control of the syringe
and allowed the depth and dose of the injection to be adjusted
electronically. The gun was positioned at an angle of 60to the
surface of the skin. In each injection, approximately 0.01 ml of
solution was injected into the skin at a depth of 2 mm using the
nappage technique in a horizontal direction; the total amount of
solution injected per session on both sides of the face was
2 ml.
The clinical status of the skin in terms of wrinkles, tautness,
and texture was evaluated and rated by the volunteer, two
dermatologists, and two independent observers before
treatment and at three and six months after the start of
treatment. Rating was based on a 5-point scale on which
improvement was rated as none (0%), mild (1–25%), moderate
(26–50%), good (51–75%), or very good (76–100%).
Punch biopsies (3 mm) were obtained from the facial skin
(treatment site) at baseline, at the end of treatment, and at
three months post-treatment. Biopsies obtained after treatment
were taken from a site near to that of the pretreatment biopsies.
Tissues were fixed in 10% buffered formalin, embedded in
paraffin, and sliced into sections 5 lm thick. All histological and
immunostaining evaluations were carried out in the Department
of Dermatology and Cutaneous Biology, Thomas Jefferson
University, Philadelphia, PA, USA.
Histochemical staining
Specimens were stained for standard hematoxylin and eosin
(H&E), Verhoeff–van Gieson (elastic fibers) (HT25A; Sigma-
Aldrich Corp., St Louis, MO, USA) and picrosirius red staining
(Direct Red 80; Sigma-Aldrich Corp.) for newly synthesized
collagen.
13
Histological measurements
Histological measurements were performed on standard H&E
stained sections using computer-based software (Image-Pro
Plus 6.1; Media Cybernetics, Inc., Silver Spring, MD, USA).
Epidermal thickness was measured from the top of the granular
cell layer to the dermo–epidermal junction. Five measurements
were calculated for each section.
Immunohistochemical staining
We evaluated collagen types I and III, as well as total elastin,
using the immunoperoxidase technique. Following antigen
retrieval by the microwave method in 0.1 Msodium citrate
(pH 6.0) for five minutes, nonspecific sites were blocked using
5% normal goat serum (NGS), 1% bovine serum albumin
(BSA), and 0.02% Triton X-100 (TX-100) in phosphate buffered
saline (PBS). Tissues were incubated at 4 C overnight with
antibodies to type I collagen (1 : 400) (sc-59772; Santa Cruz
Biotechnology, Inc., Santa Cruz, CA, USA), type III collagen
(1 : 600) (ab6310; Abcam, Inc., Cambridge, MA, USA), and
total elastin (1 : 300) (E4013; Sigma-Aldrich Corp.). Tissues
were developed using biotinylated secondary antibody (1 : 200)
(PK-6102; Vector Laboratories, Inc., Burlingame, CA, USA),
ABC reagent (Vectastain Elite ABC Peroxidase Kits Mouse,
International Journal of Dermatology 2012, 51, 913–919 ª2012 The International Society of Dermatology
Report Efficacy of mesotherapy in skin rejuvenation El-Domyati et al.
914

PK-6102; Vector Laboratories, Inc.) and DAB Chromogen
Substrate Kit (K3468; Dako North America, Inc., Carpinteria,
CA, USA), and were counterstained with hematoxylin (7211;
Fisher Thermo Scientific, Inc., Waltham, MA, USA).
Indirect immunofluorescence (IF) staining was used to detect
collagen type VII and tropoelastin. After antigen retrieval and
blocking, specimens were incubated with antibodies to type VII
collagen (1 : 600) (sc-33710; Santa Cruz Biotechnology, Inc.),
secondary antibody Alexa Fluor

goat anti-mouse IgG 594
(1 : 400) (Molecular Probes, Inc., Eugene, OR, USA) and
4¢,6-diamidino-2-phenylindole dihydrochloride (DAPI) (1 : 1000)
(D8417; Sigma-Aldrich Corp.) for nuclear staining.
The microprobe system (FD-188-10A; Fisher Thermo
Scientific, Inc.) was used to detect tropoelastin using reagents
obtained from Open Biosystems, Inc. (Huntsville, AL, USA) as
previously described.
14
Antibodies used were tropoelastin
GA317 (1 : 400) (Elastin Products Co. Inc., Owensville, MO,
USA) and Alexa Fluor

goat anti-rabbit IgG 594 (1 : 400)
(Molecular Probes, Inc.). Tissues were incubated with DAPI for
1–2 minutes.
Quantitative evaluation and statistical analysis
Quantitative evaluation of picrosirius red stained and
immunostained tissues was carried out using Image-Pro Plus
6.1; all values were normalized to baseline values. A Nikon
microscope equipped with filters to provide circularly polarized
illumination was used to evaluate picrosirius red stained tissues.
Data were tabulated and analyzed using SPSS Version 16
(SPSS, Inc., Chicago, IL, USA). Statistical analysis was
performed using Wilcoxon matched-pairs signed ranks and chi-
squared tests. Data were expressed as the mean ± standard
deviation (SD). Statistical significance was defined as P£0.05.
Results
Clinical evaluation
All six volunteers completed the mesotherapy treatment
and follow-up period. No side effects were observed.
Two of the six subjects showed mild changes (after the
third session) in terms of increased glowing of the skin,
but none reported improvement in skin tightening or
wrinkles. Clinical evaluations did not show any statisti-
cally significant effect on skin texture, tightening, or wrin-
kles in response to mesotherapy injection (Fig. 1). Results
obtained from a structured questionnaire were tabulated
and compared with data obtained at baseline to evaluate
the statistical significance of any differences using Pear-
son’s chi-squared test. Subjects showed a 10–15%
improvement in skin tightening (P= 0.06) and skin tex-
ture (P= 0.09) at the end of treatment versus a 5–10%
improvement in skin tightening (P= 0.7) and skin texture
(P= 0.8) at three months post-treatment, compared with
baseline. Clinical improvement in wrinkles ranged from
none to mild (0–5%) at the end of treatment and at
three months post-treatment compared with baseline
(P= 0.621).
Histometric changes
Microscopic evaluation of H&E stained sections revealed
no difference in epidermal thickness (64.1 ± 2.2 lm
before treatment, 67.0 ± 2.8 lm at the end of treatment,
63.3 ± 3.3 lm at three months post-treatment; P= 0.07
and P= 0.29, respectively).
Histological changes
It has been proposed that skin rejuvenation might be pro-
moted by intradermal injection of multivitamins, which
stimulate fibroblasts to produce more collagen and elas-
tin. In addition, hyaluronic acid injection is supposed to
facilitate fibroblast activation and extracellular matrix
remodeling.
9,15
We examined the effects of mesotherapy
treatment on collagen types I and III by measuring the
percentage of the dermis occupied by immunohistochemi-
cally detectable collagen and compared these values with
measurements obtained at baseline to assess the statistical
significance of any changes.
Immunoperoxidase staining for collagen type I did not
show significant differences between values obtained
before treatment (68.7 ± 5.6%) and those obtained at the
end of treatment (69.6 ± 4.9%) and at three months post-
treatment (69.1 ± 4.6%) (P= 0.765 and P= 0.889,
respectively) (Table 1, Fig. 2a). Evaluation of type III col-
lagen showed a slight increase to 65.0 ± 4.7% at the end
of treatment (P= 0.615) and 64.0 ± 4.6% at three
(a) (b) (c)
Figure 1 Clinical evaluation of a volunteer’s response to mesotherapy injection. Photographs of the periorbital area show no
significant changes in skin from (a) baseline to (b) the end of treatment or (c) 3 months post-treatment
ª2012 The International Society of Dermatology International Journal of Dermatology 2012, 51, 913–919
El-Domyati et al. Efficacy of mesotherapy in skin rejuvenation Report 915

Table 1 Outcomes in six volunteers undergoing treatment with mesotherapy administered over 3 months
Parameter
Dermis positive, %, mean ± SD P-value
Baseline
End of
treatment
3 months
post-treatment
Baseline vs.
end of
treatment
End of
treatment vs. 3 months
post-treatment
Baseline vs.
3 months
post-treatment
Collagen type I 68.7 ± 5.6 69.6 ± 4.9 69.1 ± 4.6 0.765 0.855 0.889
Collagen type III 63.3 ± 6.1 65.0 ± 4.7 64.0 ± 4.6 0.615 0.738 0.829
Collagen type VII 9.8 ± 1.1 10.2 ± 1.2 9.9 ± 1.3 0.644 0.680 0.949
Newly synthesized collagen 14.7 ± 2.1 16.6 ± 1.7 15.5 ± 2.4 0.104 0.360 0.575
Total elastin 60.7 ± 4.0 55.1 ± 3.6 58.0 ± 5.3 0.071 0.290 0.357
SD, standard deviation.
(a)
(b)
(c)
(e)
(d)
Figure 2 Dermal collagen content after mesotherapy treatment. Immunoperoxidase staining of skin specimens for collagen (a)
type I and (b) type III shows no change in collagen content. Representative samples of skin stained with picrosirius red and
viewed under a (c) bright or (d) polarized field show no noticeable change in yellow or red colors. (e) Collagen type VII expres-
sion by immunofluorescence shows no change in response to mesotherapy injection. Nuclei stained in blue with DAPI (original
magnification ·200)
International Journal of Dermatology 2012, 51, 913–919 ª2012 The International Society of Dermatology
Report Efficacy of mesotherapy in skin rejuvenation El-Domyati et al.
916

months post-treatment (P= 0.829) compared with the
baseline value of 63.3 ± 6.1%, although the changes were
not statistically significant (Table 1, Fig. 2b).
Newly synthesized collagen can be detected by picrosiri-
us red staining under polarized microscopy: large fibers
stain red and thinner fibers, which represent newly synthe-
sized fibers, show a yellow–orange stain.
13,16
Mesotherapy
injection did not appear to have any noticeable effect on
collagen formation as picrosirius red staining revealed no
significant difference in newly synthesized collagen before
treatment (14.7 ± 2.1%) compared with after treatment
(16.6 ± 1.7%; P= 0.104) and at three months post-treat-
ment (15.5 ± 2.4; P= 0.575) (Table 1, Fig. 2c,d).
Type VII collagen is the main component of anchoring
fibrils and is synthesized by both fibroblasts and keratino-
cytes; it mediates the dermo–epidermal adherence in
human skin.
17
The effect of the aging process on collagen
VII biosynthesis and degradation has been previously
noted.
18,19
Quantitative evaluation of type VII collagen
did not reveal statistically significant differences between
levels at baseline (9.8 ± 1.1%) and at the end of treat-
ment (10.2 ± 1.2%; P= 0.644) or at three months post-
treatment (9.9 ± 1.3%; P= 0.949) (Table 1, Fig. 2e).
Elastic fibers, which are responsible for the elasticity
and resilience of normal human skin, constitute <2–4%
of the extracellular matrix.
14,20,21
The accumulation of
thickened, tangled, and amorphous elastic structures in
the dermis is known as solar elastosis and represents the
histopathological mark of skin aging.
21–24
The effect of
mesotherapy injection on total dermal elastin was exam-
ined by measuring the percentage area of dermis occupied
by immunohistochemically detectable elastin; values were
then normalized to baseline. Our data revealed a statisti-
cally insignificant decrease in the level of total elastin
expression from 60.7 ± 4.0% at baseline to 55.1 ± 3.6%
at the end of treatment (P= 0.07). This was followed by
a slight, but statistically insignificant, increase in elastin
level to 58.0 ± 5.3% at three months post-treatment
(P= 0.290) (Table 1, Fig. 3a). All tissues were also
stained with Verhoeff–van Gieson special stain to differ-
entiate elastic fibers (blue–black to black) from collagen
fibers (red); this stain showed no changes in response to
treatment.
Elastic fibers are composed mainly of elastin, which is
initially synthesized as tropoelastin.
4,14
The biosynthetic
rate of elastin was evaluated by quantifying newly synthe-
sized tropoelastin. Mesotherapy injection did not appear
to bring about any significant differences between tropo-
elastin levels prior to treatment (13.6 ± 1.3%) and levels
at the end of treatment (13.9 ± 3.3%; P= 0.636) or at
three months post-treatment (13.0 ± 1.9%; P= 0.586)
(Table 1, Fig. 3b,c).
(a)
(b)
(c)
Figure 3 Dermal elastin in response to mesotherapy treatment. (a) Immunoperoxidase staining of skin tissues for total elastin
shows no significant change in levels at the end of treatment or at 3 months post-treatment. (b) Immunofluorescence staining of
tropoelastin shows no effect of treatment on newly synthesized tropoelastin in the dermis. (c) The same tissues were counter-
stained for nuclei (blue) with DAPI (original magnification ·200)
ª2012 The International Society of Dermatology International Journal of Dermatology 2012, 51, 913–919
El-Domyati et al. Efficacy of mesotherapy in skin rejuvenation Report 917

Discussion
Mesotherapy, which derives its name from the Greek
words ‘‘mesos’’ (middle) and ‘‘therapeia’’ (to treat medi-
cally), was originally developed in Europe, is one of the
newest techniques in cosmetic medicine proposed to
rejuvenate aging skin, and can be performed by medical
and non-medical professionals.
6,11
Although the US
Food and Drug Administration (FDA) has approved
most of the ingredients used in mesotherapy injection,
these components are being applied for indications for
which they currently unapproved.
6,25,26
The efficacy,
treatment protocols, pharmacokinetics, and safety of
mesotherapy are still of concern and under debate.
8,27
Improvements in wrinkles, increased elasticity, and
enhanced skin texture have been attributed to mesother-
apy injection but have not been rigorously pro-
ven.
8,10,28,29
At present, the basic concerns pertaining to
the mesotherapy approach to skin rejuvenation are that
peer-reviewed publications dealing with the efficacy of
mesotherapy are few, and published evidence-based sci-
entific studies do not support the treatment rationale or
address the issue of whether the effects, if any, are tem-
porary or permanent.
10
To the best of our knowledge,
no previous work concerning objective quantitative
changes in newly formed collagen (using picrosirius red
stain), collagen types I, III and VII, elastin, and tropoe-
lastin (using immunohistochemistry) after mesotherapy
skin rejuvenation has been presented.
In the present study, we objectively quantified both the
clinical and histological effects of mesotherapy on photo-
aging and wrinkle reduction. The evaluation of our vol-
unteers revealed no clinical improvement, and the
histological effects were statistically insignificant.
Although some improvement was seen in the skin texture
of two subjects after the third session, most volunteers
were unimpressed with the results throughout the treat-
ment and follow-up period.
The results of the present work are consistent with
those of a previous study
8
which reported that mesother-
apy injection did not result in any photographically dis-
cernible differences between treated subjects and controls,
and no significant clinical or histological changes in colla-
gen fibers were detected using H&E staining and electron
microscopy. Moreover, in the present study, we tried to
strengthen the subjective evaluation by quantitatively
evaluating changes in collagen and elastin using immuno-
histochemistry.
It is also important to point out that the American
Society of Plastic Surgeons has not approved the use of
injectable mesotherapy treatments for any indication but
has recommended that further research into the safety
and efficacy of such treatments is required.
28
By contrast, Lacarrubba et al.
9
treated 20 women with
signs of photoaging on the dorsum of the hands with
hyaluronic acid salts (of biotechnological origin) and mul-
tivitamin injections (weekly for four weeks). The authors,
using ultrasonography, reported that 15 of 19 patients
who completed the study showed an increase in the sub-
epidermal low-echogenic band (SLEB) and concluded that
mesotherapy may be an effective treatment for skin
photoaging.
9
However, Sandby-Moller and Wulf reported
that the SLEB layer was not detected in children, whereas
an increase in the SLEB layer was observed in over 50%
of adults aged >40 years. The age-related increase in the
SLEB layer was attributed to chronic ultraviolet B (UVB)
exposure.
30
Thus, Lacarrubba et al.
9
may have errone-
ously concluded that mesotherapy may be an effective
treatment for skin photoaging. Furthermore, these authors
did not confirm their clinical data by histological evalua-
tion of the treated area.
Conclusions
As with any new procedure, it is important to assess the
benefits, safety, efficacy, and standardization of mesother-
apy before it can be advocated for skin rejuvenation treat-
ments. In the present study, although mild clinical
improvement in skin texture was observed in some study
participants (n= 2) after the third session, subjective, as
well as objective, quantitative evaluation suggested that
mesotherapy treatment utilizing the protocol established
in this study has no significant beneficial effect in reversing
the signs of skin aging, either clinically or histologically.
However, further in-depth, long-term studies on a large
cohort of patients are required to identify the maximum
duration of treatment necessary to achieve clinical or his-
tological benefits if, indeed, these are to be obtained.
Acknowledgments
The authors would like to thank the Cultural and Educa-
tional Bureau of Egypt for its financial support of this
work. We also thank Alicia Dowling for critically review-
ing this manuscript.
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