JOURNAL OF COSMETIC AND LASER THERAPY

http://dx.doi.org/10.1080/14764172.2016.1247962

REPORT

Confocal microscopy and imaging profilometry: A new tool aimed to evaluate

aesthetic procedures
Gabriella Fabbrocinia, Caterina Mazzellaa, Fabio Montagnarob, Maria Pia De Padovac, Sandra Lorenzid, Aurora Tedeschie,
Patrizia Forgionef, Claudia Capassoa, Luigi Siverog, Carla Velottih, Daniela Russoi, Rosa Vitiellob, and Gennaro Ilardii
aDepartment of Clinical Medicine and Surgery, Section of Dermatology, University of Naples Federico II, Italy; bDepartment of Chemical Sciences,
University of Naples Federico II, Italy; cDermatology, Nigrisoli Private Hospital, Bologna, Italy; dDepartment of Dermatology, University of Bologna,
Italy; eDermatology Clinic, University of Catania, Italy; fDermatology Clinic, Ascalesi Hospital, Naples, Italy; gDepartment of Advanced Biomedical
Sciences, University of Naples Federico II, Italy; hDepartment of Chemical Engineering, Materials and Industrial Production, University of Naples
Federico II, Italy; iDepartment of Advanced Biomedical Sciences, Pathology Section, University of Naples Federico II, Italy

ABSTRACT ARTICLE HISTORY

According to the American Academy of Aesthetic Plastic Surgeons, more than 11 million cosmetic surgical Received 21 February 2016
and nonsurgical procedures were performed by board-certified plastic surgeons, dermatologists and Accepted 9 October 2016
otolaryngologists in the United States, totaling more than 12 billion dollars. We performed a retrospective KEYWORDS
observational multi-centric study on patients treated with a non-animal origin cross-linked hyaluronic Confocal microscopy;
acid with different molecular weights for nasolabial folds, evaluating through a new imaging system, hyaluronic acid fillers;
profilometric techniques with the confocal microscopy, the durability, the efficacy and the safety of profilometry
this product. From 25 patients, 150 silicone casts were obtained: 75 casts of the right nasolabial fold and
75 casts of the left nasolabial fold. Roughness arithmetical average of the right fold at T2 decreased by 50%
versus T0 and by 40% compared to T1; at T2, it decreased by the 45% versus T0 and by 35% compared to
T1. No side effects were reported. Results proved that the analysis of the skin microreliefs through confocal
microscopy is a new imaging system that allows to evaluate with precision and safety the results of aesthetic
treatments such as fillers objectively.
Abbreviations: (HA) Hyaluronic Acid, (CLHA) Cross-Linked HA, (Ra) arithmetical average roughness, (GAIS)
Global Aesthetic Improvement Scale

Introduction of matrix metalloproteinases (TIMPs) further act to retard

The dermal extracellular matrix is composed of a dense
network of interlinked collagen and elastic fibers, contain-
ing water-retaining proteoglycans and glycosaminoglycans,
accounting for their biomechanical and viscoelastic properties
(1). Procollagen is secreted from fibroblasts, and the peptide
ends are removed by two enzymes in the extracellular space.
Removal of the ends produces collagen, which spontaneously
assembles into large fibers that are enzymatically cross-linked.
This cross-linking is necessary for normal structural support.
Type I collagen undergoes natural breakdown by enzymatic
degradation; however, this degradation in human skin is
exceedingly slow. Humans express only four enzymes that are
capable of initiating breakdown of type I collagen. These colla-
genases are members of a family of matrix protein-degrading
enzymes, referred to as matrix metalloproteinases (MMPs).
MMPs are responsible for physiological degradation of vari-
ous extracellular matrix proteins. In addition, skin expresses
natural inhibitors of these MMPs. These tissue inhibitors
collagen breakdown. Thus, type I collagen in human skin is
very stable, requiring approximately 30 years on average to
undergo replacement.
This slow rate of type I collagen turnover allows accumula-
tion of age-dependent modifications that impair its functions.
These alterations include formation of new cross-links derived
from sugars. Importantly, these crosslinks are not able to be
efficiently broken down and removed during the slow normal
process of MMP-mediated turnover, causing accumulation of
fragmented collagen within the extracellular matrix as skin
ages. Cross-links prevent complete removal of collagen frag-
ments. The fragments cannot be repaired or incorporated into
newly made collagen fibrils and therefore cause defects in the
three-dimensional collagen matrix. These defects impair the
structural and mechanical integrity of the dermis and thereby
deleteriously alter its function. Accumulation of fragmented
collagen lies at the heart of age-related changes in the appearance
of human skin.

CONTACT Gabriella Fabbrocini gafabbro@unina.it Department of Clinical Medicine and Surgery, Section of Dermatology, University of Naples Federico II,
Via Pansini 5, 80131 Naples, Italy.
Color versions of one or more of the figures in the article can be found online at http://www.tandfonline.com/ijcl.
© 2016 Taylor & Francis Group, LLC
2 G. FABBROCINI ET AL.
UV irradiation acutely induces collagen-degrading MMP
activities and suppresses collagen production. Thus, UV irra-
diation, like chronological aging, shifts the balance toward net
collagen degradation (2).
Consequently, several fillers for soft-tissue augmentation
procedures including treatment of nasolabial (3) perioral (4)
and periorbital wrinkles (5) have been developed. Hyaluronic
acid (HA) dermal fillers have been extensively used for skin
rejuvenation procedures in aesthetic medicine (6). It is very dif-
ficult to establish the best filler for the best site. Some diagnostic
procedures can be used.
HA is a glycosaminoglycan consisting of alternate repeating
D-glucuronic acid and N-acetylglucosamine units and is dis-
tributed throughout the extracellular matrix, connective tissues
and organs of all higher animals (7). HA enhances hydration in
the extracellular space due to its ability to capture water mol-
ecules and to induce optimal physiological conditions in the
extracellular matrix for proliferation and organization of der-
mal cells (8). HA, used as a filler, can have an avian or a bacterial
origin with the latter being largely preferred for its high purity,
increased viscosity and decreased potential to induce allergic
reactions (9).
Analysis of elastic fibers and collagen in the skin is a reliable
tool to assess the efficacy of new HA-based compounds used
for rejuvenation procedures. In fact, injection of cross-linked
HA (CLHA) into dermal equivalent cultures results in elonga-
tion of fibroblasts together with type I collagen synthesis due
to upregulation of transforming growth factor beta (TGF-E)
pathway (10). Furthermore, HA injection significantly increases
the synthesis of neo-collagen I and pro-collagen I and III
mRNA compared to baseline, as observed 30 days post admin-
istration (11). CLHA injection into the buttocks skin of subjects
aged over 70 years stimulated fibroblasts to ultimately produce
type I collagen (10).
HA fillers can be distinguished on the basis of the particle
size, cross-linking agent used, cross-linking degree, percentage
of cross-linked HA and amount of free (unmodified) HA.
Confocal microscopy and imaging profilometry can be
important tools to evaluate the efficacy of some aesthetic pro-
cedures. The aim of the study was to evaluate the efficacy of this
new technique as a diagnostic procedure to test the durability,
the validity and the safety of a cross-linked HA of non-animal
origin with different molecular weights for the treatment of
nasolabial fold.
Material and methods
All participants signed informed consent prior to treatment.
All procedures conformed to the guidelines set forth by the
Declaration of Helsinki.
The tested product, used in order to perform the treat-
ment, was HA (25 mg/ml, 1000–2000 kDa, 23  3 Newton [N]
extrusion force, 1,4-Butanediol Diglycidyl Ether (BDDE) con-
tent 0.1 ppm). It is commercially available with the trade-
mark Aliaxin£ GP (Global Performance) and distributed by
IBSA Farmaceutici Italia S.r.l. Six different dermatologists in
Naples, Bologna and Catania participated in this retrospective
multicentric observational study and performed the treatment
with injection of tested product. 25 patients (25 women), aged
47.6  1.3 (range: 40–60) years were involved. The inclusion
criterion for this study was the clinical evidence of moderate-
to-severe wrinkles affecting the nasolabial folds, with a degree
of photoaging level III according to Rubin’s scale or type
III according to Glogau’s scale. Exclusion criteria were his-
tory of severe allergic disorders, cutaneous disease affecting
the face such as acne and herpes, known hypersensitivity to
HA, history of autoimmune disease, abnormal liver function,
pregnancy, lactation and history of skin cancer; use of antico-
agulant therapy such as aspirin or warfarin, use of topical ste-
roids, retinoid medications, or anti-acne agents in the 4 weeks
previous the treatment; microdermabrasion, chemical peels
or use of fillers in the 24 weeks before the treatment. Patients
received an injection of 0.5 ml of the product for each naso-
labial fold requiring the anti-aging treatment. Injections were
performed with a linear retrograde technique. Once the pro-
cedure was completed, the injection site was manually mas-
saged to allow the implanted material to adapt to the adjacent
tissues. Visits were planned at 4 months (T1) and 6 months
(T2) after the treatment.
Patients were photographed before and after the injections
and at T1 and T2, in order to evaluate the aesthetic result,
comparing the clinical images before treatment, immedi-
ately after, and at the time T1 and T2. Rather to assess, in the
most objective way, the durability of the product beginning
from the first injection until the last T2 control, we used
casts of nasolabial folds obtained using a silicone material.
Finally, the silicon casts were analyzed through confocal
microscopy and profilometry. In this manner, we can appre-
ciate the aesthetic result by the photos and the durability of
the treatment through confocal microscopy and imaging
profilometry.
Overall 150 silicone casts were collected: 75 of the right
nasolabial fold and 75 of the left nasolabial fold.
The analysis of the skin microreliefs through confocal
microscopy and profilometry
The silicone casts of nasolabial folds collected as previously
described were analyzed with the aid of a confocal micro-
scope, to assess the surface roughness and morphology of the
folds. Leica Scan DCM - 3D was used for the microgeomet-
ric characterization together with the two dedicated software
systems: LeicaScan and Leica Map, respectively, to manage
the different acquisition modes and options and for the image
post-acquisition processing and analysis.
The main operating conditions were: optical magnification:
5; optical resolution: 0.94 microns; lens numerical aperture:
0.15; lens field of view: 2550  1910 microns. This microscope
is able to acquire an extended area of a given surface. In the
present case, this area was set equal to the lens view field. The
value of the z-scan (i.e., the acquisition shift along the direc-
tion normal to the acquired area) was set at 1.8 mm. Once an
image is acquired, the dedicated software post-processes it by
automatic leveling, and by the application of a Gaussian filter
to exclude the so-called “waviness” effect on the roughness
quantitative determination.
All the acquired images were similarly processed: empty
areas were reconstructed by choosing the proper descriptive
 JOURNAL OF COSMETIC AND LASER THERAPY 3

algorithm, and the irregularities were flattened using the least

square regression method. These images were treated by the
software as optical sections and combined in order to reconstruct
a 3-D image of the analysed samples.
The final 3-D images were analyzed to obtain the roughness
profile of the fold and to calculate Ra, the arithmetical average
roughness. Ra is the arithmetical average of the absolute values
of the deviations profile, within the length of the base L, as
generally depicted in Figure 1, where the formula used for the
determination of Ra is also reported (Figure 1). The collected

Figure 1. Ra is the arithmetic average of the absolute values of the deviations

profile within the length of the base (L).

Figure 3. Patient Y. 3D images of surface wrinkles acquired by confocal micro-

Figure 2. Patient X. 3D view of the fold surface acquired by confocal microscope; a) scope; (a) immediately after injection of the study product (T0), (b) four months
immediately after injection of the study product (T0), b) 4 months after injection of after injection of the study product (T1), and (c) six months after injection of the
the study product (T1), and c) 6 months after injection of the study product (T2). study product (T2).
4 G. FABBROCINI ET AL.

Figure 4. Patient X. (a) Nasolabial folds of the patient before injection. (b) Aesthetic results immediately after injection of the study product (T0). (c) Aesthetic results 4
months after injection of the study product (T1). (d) Aesthetic results 6 months after injection of the study product (T2).

data were used to evaluate the roughness associated to the folds
after the treatment at T1 and T2 (Figures 2, 3).
Results
In this study, all patients demonstrated aesthetic improve-
ments well documented by the photographs taken before and
immediately after injection of the tested product. Moreover,
the photographs collected during the following visits showed a
good durability of the aesthetic correction of the nasolabial
folds even six months after the treatment (Figures 4, 5a–d).
Combining the profilometric techniques with the confo-
cal microscopy, we obtained an objective way to measure the
durability of the aesthetic correction: Ra of the right fold at T2

Figure 5. Patient Y. (a) Nasolabial folds of the patient before injection. (b) Aesthetic results immediately after injection of the study product (T0). (c) Aesthetic results four
months after injection of the study product (T1). (d) Aesthetic results at six months after injection of the study product (T2).

decreased by the 50% versus T0 and by 40% compared to T1; Ra
at T2 decreased by the 45% versus T0 and by 35% compared to
T1. 80% of the patients showed “exceptional improvement” at
T1, as the evaluation with the Global Aesthetic Improvement
Scale showed, and this result did not change at T2.
All the patients were satisfied immediately after the
procedure, and no side effects were reported during the obser-
vation period beyond mild symptoms (pain, sensation of heat,
reddening in the injection site) also described by the product
technical sheet.
Discussion
Silicone skin casts of the treated area were collected in this
study immediately after the treatment (T0) and at T1 and T2 of
nasolabial folds with a cross-linked HA of non-animal origin
with a combination of different molecular weights.
Generally, the acquisition of the skin cast images is performed
by a stereomicroscope connected to an analogic camera, and
the study of the morphometric skin surface is used to evalu-
ate the regularity of the skin texture surface and the changes
of this aspect after filler treatments (12,13). The images of
the replicas are then subject to the study of the Fourier spec-
trum, able to identify the mathematical descriptor parameters
of the skin regularity degree in an objective and reproducible
way. Moreover, the Fourier transform of the skin texture sur-
face image is evaluated by the mean gray values obtained along
the x-axis and the y-axis: these averages are calculated from
the parameter descriptors. A number of studies have been
performed to evaluate the durability and safety of fillers using
this methodology (12,13).
For the first time, profilometric techniques on skin casts were
combined with confocal microscopy. Confocal profilometry is
a method that ensures reproducibility and comparability of the
data, with standardized methods of acquisition. It produces very
high-quality 3D images through virtual confocal image planes.
The images obtained are similar to those obtained with the 3D
reconstruction through magnetic resonance and through com-
puted tomography. The key point of the confocal technique is
the removal of interference between adjacent planes by the use
of so-called pinhole, a tiny camera perforation that allows the
instrument to acquire the images.
This new non-invasive approach could be used as a guide
for aesthetic physicians in the choice of different products, for
different patients, in order to optimize the choice and to better
respond to the patient’s expectations. In aesthetic medicine, it
would be really useful to assess the efficacy and durability of
the dermal fillers present in the market. Aesthetic physician
community should ask for these kind of investigations in order
JOURNAL OF COSMETIC AND LASER THERAPY 5
to set appropriate guidelines, enabling them the possibility to
guarantee patient safety and satisfaction while avoiding, as
much as possible, side effects.
Declaration of interest
The authors state no conflict of interest.
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