The role of hyaluronic acid and amino acid against the aging of the

Accepted Article
human skin: a clinical and histological study.

Running title: biomodulation of the skin

A Scarano6*, A. Sbarbati2, R Amore3, E.L. Iorio4, G. Ferraro5, M. Marchetti6 and D. Amuso7

1Dean of Master course in Aesthetic Medicine, Department of Medical, Oral and Biotechnological
Sciences, University of Chieti-Pescara, Italy; e.mail. ascarano@unich.it; tel. +39 08713554084;
fax. +3908713554099

2University of Palermo, Master of Techniques of Aesthetic Medicine and Wellness, Palermo,

Italy; e.mail. andrea.sbarbati@univr.it

3Department of Neurosciences, Biomedicine and Movement Sciences, Anatomy and Histology

Section, University of Verona, School of Medicine, Verona, Italy; e.mail:
robertoamore@hotmail.com

4Master course in Aesthetic Medicine, Department of Medical, Oral and Biotechnological

Sciences, University of Chieti-Pescara, Italy. eugenioluigi.iorio@gmail.com

5Department of Plastic, Reconstructive and Aesthetic Surgery, Università degli Studi della
Campania Luigi Vanvitelli, Naples, Italy. e.mail: giuseppe.ferraro@unicampania.it

6Ph.D School of Applied Medical-Surgical Sciences, University of Rome Tor Vergata, Rome,
Italy marco@marcomarchetti.it

7University of Palermo, Master of Techniques of Aesthetic Medicine and Wellness, Palermo,

Italy; e.mail. d.amuso.estetica@gmail.com

* Correspondence: Prof. Antonio Scarano; Department of Medical, Oral and

Biotechnological Sciences, University of Chieti-Pescara, Via dei Vestini 31, 66100 Chieti, Italy.
Email: ascarano@unich.it.

Running Head: biomodulation of the skin

This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/JOCD.13811
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Accepted Article
Author Contributions: The following statements should be used “Conceptualization, A.Sc. and
D.A.; methodology, D.A., E.L.I. and G.F.; software, R.A. and M.M; validation, F.L., and A.Sb.;
formal analysis, A.Sc.; investigation, A.Sc., D.A., R.A., G.F. and M.M. ; resources, A.Sc. data
curation, D.A.; writing—original draft preparation, A.Sc.; writing—review and editing, A.SC.,
D.A.,A.Sb., R.A., G.F. and E.L.I., visualization, A.Sc.; supervision, A.Sc.; project administration,
A.Sc.; funding acquisition, A.Sc. All authors have read and agreed to the published version of the
manuscript.

This article is protected by copyright. All rights reserved

Accepted Article
DR. ANTONIO SCARANO (Orcid ID : 0000-0003-1374-6146)

Article type : Original Contribution

The role of hyaluronic acid and amino acid against the aging of
the human skin: a clinical and histological study.

Running Head: biomodulation of the skin

Abstract

Background: In aesthetic medicine different techniques have been used against the aging of
the human skin especially in the facial area. Hyaluronic acid is used for improving the
quantity of water and extracellular matrix molecule. The aim of this study is a clinical and
histological evaluation of the effect of low molecular weight hyaluronic acid fragments
mixed with amino acid (HAAM) on the rejuvenation the face skin treated with intradermal
microinjections. Methods: Twenty women with mean age 45 between range 35-64 were
studied, there of 8 in menopause and 12 of childbearing age. The patients were treated with
the HAAM products by mesotherapy technique; before and after three months of the
therapeutic procedure each patient underwent small biopsies with a circular punch biopsy.

Results: The clinical results of the present study showed, that the administration of the
dermal filler containing fragments of hyaluronic acid between 20 and 38 monomers and
amino acid via dermis injection technique produces an aesthetic improvement in the faces of
the treated patients, while the histological evaluation shows an increased fibroblast activity
with the production of Type III reticular collagen and increased number of vessels and
epidermis thickness. Conclusions: The clinical and histological assessment showed that

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 subcutaneous HAAM infiltration have a significant impact on the dermis and clinical aspects
Accepted Article
of the face.

Keywords: skin aging, biomodulation, hyaluronic acid fragments, amino acid.

1. Introduction

Human skin, together with its undeniable protective role, performs the delicate task of
mediating the metabolic and information exchanges between the body and the environment
around it. Unfortunately, due to a series of exogenous (e.g. ultraviolet radiation) and/or
endogenous stressors (e.g. endocrine-metabolic diseases) and aided by the inexorable flow of
time, it may undergo a sequence of functional alterations and/or structural problems that
could prejudice its integrity more or less seriously1. This leads to dyschromia, dehydration,
loss of elasticity, microvascular changes, etc. which may evolve into a serious aesthetic
problem if they are found in critical areas, such as face or décolleté2,3. For the prevention and
treatment of the resulting skin imperfections - first of all wrinkles, but not only these – the
approach of "cutaneous biostimulation" has been proposed for some time by cosmetic
procedures, based on an injective technique, having the primary purpose to regenerate the
normal structure and functions of the skin by a targeted action on the fibroblasts that compose
it, which means, on the surrounding extracellular matrix4.

Actually, the fibroblast-myofibroblast-fibrocyte complex is only one element in a much

larger chessboard, which includes other components, both cellular (endothelium of blood and
lymphatic vessels, monocyte-macrophages, lymphocytes, nerve ganglia, etc.) and non-
cellular (extracellular matrix, and others) and their anabolic processes must be continually
balanced by catabolic processes and by cellular reprocessing, under the control of signal
molecules (from free radicals to cytokines and up to growth factors)5,6. In other words, given
the structural and functional complexity of the skin at molecular level, it is clear that
fibroblast stimulation alone may be inadequate to prevent or treat the imperfections without
an "integrated" approach. In the field of aesthetic medicine, an important fact is that
fragments of hyaluronic acid between 20 and 30 monomers activate the fibroblasts by
binding to CD44 receptors and thus the production of reticular collagen type III7,8. This

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 phenomenon, of course, is subordinated to the bioavailability of the precursors, enzymes and
Accepted Article
cofactors involved in the biosynthesis of this structural protein, as specified below9,10.

In this study the role of hyaluronic acid and some organic and inorganic substances was
considered in the prevention and the treatment of some common skin imperfections. The
purpose of this study is a clinical and histological evaluation of the effect of low molecular
weight hyaluronic acid fragments and amino acid (HAAM) on the rejuvenation of face skin
treated with intradermal microinjections.

2. Materials and Methods

2.1 Inclusion and Exclusion Criteria

Twenty women with mean age 45, range 35-64, were studied, thereof 8 in menopause and 12
of childbearing age. This study was conducted in the private office. The study was based in a
private multi-specialty Medical practice in Montesilvano and Modena (Italy), in full
accordance with ethical principles, including the World Medical Association Declaration of
Helsinki (https://www.wma.net/wp-content/uploads/2018/07/DoH-Oct2008.pdf) and the
additional requirements of Italian law. All patient signed informed consent on the adopted
procedure, but none of the patients knew which pathology the treatment was aimed at. All
patients presented generalized rhytidosis, poor skin hydration and reduced sebum production,
under dry, hypoxic conditions with no changes in diet during treatment. Exclusion criteria
were diet changes, pregnancy, strong smoker (20 cigarettes per day) history of allergic and/or
irritant contact hand dermatitis, systemic diseases and psychiatric illness.

2.2 Clinical evaluation

After anamnestic data collection and physical examination, the patients were submitted to
pH measurement, assessment of sebometry and hydrometry; photography and echography in
the glabella, canthal, malar-cheek, chin and neck regions. For evolving the skin hydration,
sebum and Ph, SoftPlus (Callegari, Parma, Italy) was used. Hydration was expressed in g/m2
/h while sebum µg/cm2/min. Each value was expressed average and SD.

2.3 Clinical Procedures

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 In this study a common base of low molecular weight hyaluronic acid fragments was used, to
Accepted Article
which was added, a variously buffered aqueous solution so composed: Sodium Hyaluronate
with L-lysine, L-proline, L-alanine, glycine, L-serine, L-cysteine, L-leucine, L-valine, L-
isoleucine buffer in a solution with Sodium bicarbonate (SKIN-B® Italfarmacia, Rome,
Italy). The patients were treated with the HAAM products by mesotherapy technique,
according to a protocol of 8 weeks providing a treatment on a weekly basis with the use of
2.5 cc syringes and 13 mm 30G needles. The solution was injected into the deep layer of the
dermis with a suitable amount of at least 0.2 / 0.3 mL in the cutaneous points where
hypotonia was present. We used a 2.5 mL syringe for better dosing and a meso needle 30G x
6 mm. One session every 15 days, for 4 times. The ultrasound exams were performed in B-
mode in which 2D (Esaote touch Genova Italy) and images of longitudinal or transverse
orientation were taken. Ultrasound was used at 5 to 13 Mega-hertz frequencies for
visualization of epidermis, dermis, and subcutaneous structures. Morphological
characteristics and thickness of the dermis in the skin glabella, canthal, malar-cheek, chin,
neck regions and mandibular angle were evaluated.

2.3 Histological Procedures

Before and after three months of therapeutic procedure, each patient underwent small
biopsies with a circular punch biopsy of 2 millimeters diameter (KAI Industries, Oyana
Japan). The biopsies were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH
7.4) for 24 h at 4°C, then dehydrated in a graded series of ethanol rinses and finally
embedded in paraffin and sectioned at a thickness of 4 µm. In total, 40 biopsies were
retrieved, 20 before treatment and 20 after treatment. The specimens were stained with
Haematoxylin and Eosin for general consideration, for histomorphometry evaluation
Masson’s trichrome and the Van Gieson method were utilized. Four fields of 2000 μm in
diameter to 4000 μm in length were evaluated for each sample.

A histological evaluation ad hoc was performed using HAAM on a sample of subjects with
skin imperfections. The immuno-histochemical reaction to the Epidermal Growth Factor
(EGF) was measured.

The biopsies were carried out to document:

- epithelial thickness excluding the stratum corneum.

- vessel number;

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 -cells positive for Epidermal Growth Factor (EGF)
Accepted Article
-Qualitative evaluation of type III collagen with multiple stained histology.

-Qualitative evaluation of elastic fibres with multiple stained histology.

2.4 Immunohistochemical Localization of EGF

The slides were submitted for immunohistochemistry (Avidin-Biotin-Peroxidase Complex)

using mouse monoclonal antibodies that are specifically against EGF in the dilution 1:30
(mouse monoclonal anti human epidermal growth factor, lot 3360, clone H11, Dako
Cytomation, EUA). The slides were incubated for 30 min with 100 µl of anti-EGF with 2%
goat serum (20 µg/ml) and rinsed twice in PBS and were assayed by incubating Vectastain
ABCAP Reagent for 30 min and rinsing twice in PBS. The color reaction was developed by
incubating sections for 30-60 min with Alkaline Phosphatase Substrate Solution III prepared
with µ1 mM levamisole to block endogenous peroxidase activity. If the color development
was sufficient, slides were rinsed in distilled water to stop the color reaction and were
coverslipped.

A positive reaction to the EGF antibody was considered when the color brown appeared on
the cytoplasmic membranous area of the cell.

Statistical analysis

A power analysis was performed using clinical software for determining the number of
samples needed to achieve statistical significance for quantitative analyses of cell numbers
for:

- epithelial thickness

positive Epidermal Growth Factor (EGF)

- vessel number.

2.5 Statistical Evaluation

A calculation model was adopted for dichotomous variables (yes/no effect) by using the
incidence effect designed to discern the reasons (90% for the Test group and 10% for the

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 control group), with alpha = 0.05 and power = 80%. The optimal number of samples for
Accepted Article
analysis was 10 patients per group. Numerical results are presented as the ±SD means of all
the experiments. The data outcome was collected and statistically evaluated by the software
package Graphpad 6 (Prism, San Diego CA- USA). The normal distribution of the study data
was evaluated by the Kolmogorov-Smirnov test to evaluate the normal distribution. The
Wilcoxson signed-rank test was performed to compare the study variables means in each
group. The level of significance was set at p<0.05.

thickness of the dermis in the skin glabella, canthal, malar-cheek, chin, neck regions and
mandibular angle were evaluated.

3. Results

3.1 Clinical evaluation

All the patients completed the follow up. The treatment with the injectable medical devices of
the HAAM was associated with favorable results, at a photographic level, as indicated by an
evident reduction of periorbital and frontal wrinkles, by improvement of the skin texture,
increase in skin glow and turgidity and also in volumes, which immediately make the skin
appear healthier and aesthetically more beautiful (Fig. 1) The echographic examination
showed an improvement of the dermis thickness in the glabella, canthal, malar-cheek, chin,
neck regions and angle of the mandible region with a mean improvement of 27.3% compared
with measurements obtained at the beginning of the study in the first month; this
improvement was statistically significant (p=0.007). (fig. 2, Table 1) Likewise the values of
pH-metry, sebometry and hydration compared to the baseline resulted improved (Table 1). In
fact, the goal was achieved and specifically, that of demonstrating that the administration of
the medical devices containing fragments of hyaluronic acid between 20 and 38 monomers
via injection technique produces an aesthetic improvement in the face of the treated patients.

3.2 Histology evaluation

The histological results of the slides were read by two pathologists working in two different
universities of medicine, Verona and Modena, to minimize the possibility of interpretation.

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 Before treatment: The biopsies showed loss of the rete collagen bundles to the collagen.
Accepted Article
Additionally, alteration of the microcirculation was evident before treatment. Few fibroblasts
and the epidermal with a typical aspect of some components of microcirculation were
observed. The specimens were intensely colored for collagen. No pathologic inflammatory
cells were observed in the epithelium or epidermis (figs. 3and 4). The histomorphometric
results are shown in the table 1 and fig. 5.

After treatment: These results show that these injectable HAAM are actually capable of
stimulating the fibroblast activity with the production of Type III reticular collagen. The
epidermis showed up well-structured and with increased thickness. An evident replicative
activity of the epidermis was observed with positivity for immuno-histochemical reaction to
the Epidermal Growth Factor (EGF). The fibroblasts were immunohistochemically positive
in the nuclei of the germinative layer of the epidermis (basal layer) and in some cells of the
dermis. The visible fibres of collagen appeared reorganized and the microcirculation was
present. There was a clear increase in fibre collagen in the epidermis and epidermal thickness
without pathological presence of inflammatory cells (figs. 3and 4). The histomorphometric
results are shown in the table 1 and fig. 5.

4. Discussion

The clinical results of the present showed, that the administration of the medical devices
containing fragments of hyaluronic acid between 20 and 38 monomers and amino acid via
dermis injection technique produces an aesthetic improvement in the faces of the treated
patients. The treatment with HAAM produces an impact on epidermal hydration which may
signify that more water is available for the normal physiological processes. The histological
evaluation showed an increased fibroblast activity with the production of Type III reticular
collagen. Also, it evidenced an increased number of vessels and the epidermis showed
increased thickness with augmentation of cells of the basal stratus, then an increase of EGF.
Reduction of the activity of EGF signaling is widely associated with skin aging11–13. It is
known to be one of the crucial growth factors in wound healing14 through mitosis and
chemostasis stimulation of the epithelial cells15. The visible fibres of collagen appeared
reorganized and the microcirculation was present. There was a clear increase in fibre collagen
in the epidermis and epidermal thickness without pathological presence of inflammatory
cells. Hyaluronate (HA) is extensively used in aesthetic medicine for soft tissue
16
augmentation and skin rejuvenation . It is synthesized on the cell membrane of many cells,

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 such as fibroblasts and keratinocytes and plays an important role in the hydration of medium
Accepted Article
connective tissues facilitating the cell movement in inflammation, and wound healing17 and
its decrease is associated with skin aging18. In the present study we chose the fragments of
hyaluronic acid between 20 and 38 monomers mixed with amino acids because they play an
important role in the biomodulation of the dermis19–26. Hyaluronic acid is the most common
glycosaminoglycan of the extracellular matrix and reaches its maximum concentration only
in highly hydrated tissues, such as the vitreous fluid and the umbilical cord24. It is degraded
into fragments by hyaluronidases that stimulate angiogenesis, tissue remodeling and cell
turnover25,27. The length of the hyaluronate fragments (HF) influence its biological effect.
The more receptor sites and the longer HF are exposed to them, the lower the possibility that
the ligand disconnects from the cell surface. By gradually increasing HF length, the transition
to 20-22 monomers is accompanied by a dramatic increase in receptor avidity, which is then
tripled28,29. A HF of 20 monomers, with two hexamers, spaced by an octamer (probably with
a favorable conformation, e.g. helicoidal), each of them binding to a CD44 receptor, achieves
an optimal condition30. HF equal to or higher than 30 units gives no warranty for better
performance in terms of binding31,32. Shorter HF demonstrate variable effects, related to the
modulation of the inflammatory process and of the immune response, also thanks to
simultaneous or sequential activation of other receptors, such as the receptor for Hyaluronan-
Mediated Motility (RHAMM), the Toll-like receptors (TLRs) , the Hyaluronan Receptor for
Endocytosis (HARE) and Lymphatic Vessel Endothelial Receptor 1 (LYVE-1)33. In
particular, the stimulation of RHAMM receptors promotes fibroblast mobility and activation
of macrophages34. The hyaluronic acid fragments integrated with amino acids (HAAM)
completely meet all requirements of modern physiological biomodulation, because they attain
their target to change ad-hoc the metabolic and / or information flows in order to re-establish
the morpho-functional harmony of skin suffering from imperfections, just like the
instruments of an orchestra change a sound or a tone to obtain a melodic effect35–37. On the
basis of these observations the fragments of low molecular weight hyaluronic acid, by
stimulation of the inflammatory cascade and the immune system, reduction of the expression
of the matrix metalloproteinases MMP-1 and MPP-3, the stimulation of the angiogenesis, and
the control of oxidative stress, will favor, in an acute phase, the processes of tissue
regeneration, giving to hyaluronic acid the role of real physiological modulator38. On the
basis of this clinical and histological evidence here discussed, we can assume a common
basic mechanism of the product (HAAM) in which are put together specific effects related to
hyaluronic acid and amino acid. In fact, the goal was achieved and specifically, that of

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 demonstrating that the administration of the medical devices containing fragments of
Accepted Article
hyaluronic acid between 20 and 38 monomers via injection technique produces stimulation of
fibroblasts39. These favourable results achieved are due to an improvement in skin
physiology, explicitly, to an optimization of biochemical reactions and to a deep treatment of
the skin and not simply to an aesthetic method40.

Basically, the infiltration with (HAAM) is complemented by the stimulation of fibroblasts

which produce type III collagen and, probably, multiply and differentiate thereafter into
myofibroblasts to be able to promote tissue regeneration, by their contraction, and the
recovery of the skin’s dermal turgor41. Amino acids are the building blocks of proteins and
are important for physiological turnover of tissues. Several in vivo and in vitro studies have
reported that amino acids stimulate protein synthesis42,43. The amino acids were added can
help maintain standard pH of the extracellular matrix (as specified below); moreover, it is not
excluded that they can be used for the synthesis of bioactive peptides44. In this phase, mainly
glycine, L-proline and L-alanine and, to a lesser extent, L-serine, L-lysine, L-leucine and L-
isoleucine are intensively used for the synthesis of polypeptide chains of collagen; glycine, L-
valine, L-alanine and L-proline, instead, are needed for the synthesis of elastin45.
Subsequently, the branched chain amino acids L-valine, L-leucine and L-isoleucine are
necessary to promote the synthesis of actin, a contractile protein, in myofibroblasts of
fibroblast derivation46. Serine, an amino acid, target of the post-translational
kinase/phosphatase modulation processes, is also needed as a precursor of arachidonyl-serine,
an endocannabinoid, recently discovered and capable of stimulating the repair processes of
endothelium and angiogenesis47. Finally, cysteine acts as a precursor of glutathione, the most
powerful intracellular antioxidant48,49. Its role is to modulate the action of reactive oxygen
species (ROS): its excess would result damaging for the cell repair processes50. The amino
acids were used in the present study to stimulate protein synthesis. Probably the HAAM
induces, during a first phase, a condition of pro-inflammation that is functional for the
healing process. It should be highlighted that, at the highly acid pH of the device, the amino
acids do not undergo any chemical modification but only cope with the variation of the
electrolytic properties (reassortment of the hydrogen ions and conversion into mainly
protonated forms, therefore positively charged); consequent loss of polarity could promote
their capture by the fibroblasts51. The progressive diffusion of hydrogen ions of the acid
formulation stimulates a gradual hydrolysis of the hyaluronic acid fragments. Therefore, after

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 a transitory pro-inflammatory phase, the dermal reactivity is modulated completely
Accepted Article
physiologically in an anti-inflammatory sense, contributing effectively to the tissue repair.

From a standpoint of microcirculation, an increase in both the acute and chronic perfusion
capacity of the capillary bed can be observed. This capacity could be connected, in view of
the data available in literature, to structural changes of the perivascular and preadipocyte
structures52, likely associated with modifications of functional-biochemical nature, as we
proved in histological tests.

On the basis of these observations it was theorized that the fragments of low molecular
weight hyaluronic acid, by stimulation of the inflammatory cascade and the immune system,
the reduction of the expression of the matrix metalloproteinases MMP-1 and MPP-3, the
stimulation of the angiogenesis, and the control of oxidative stress, will favor, in an acute
phase, the processes of tissue regeneration, giving to hyaluronic acid the role of real
physiological modulator. In the present study, we have not evaluated the inhibitors of
metalloproteinases (TIMPs) and matrix metalloproteinases (MMPs) and this represents the
limit of this research, in a future study it would be interesting to also evaluate the MMPs and
TIMPs. However, we have estimated the fibroblast activity, the production of Type III
reticular collagen, number of vessels and epidermis thickness before and after hyaluronic acid
mixed with amino acids treatment. The data measured in this study are sufficient for
affirming that hyaluronic acid mixed with amino acids stimulate fibroblastic activity leading
to production of new extracellular matrix. Probably a minor limitation of the present study is
only the facial regions have been included. This is the first paper to demonstrate that low HA
fragments and amino acids increase the change in collagen protein with histological changes.
In conclusion the HAAM via injection technique produces stimulation of fibroblasts and
aesthetic improvement in the faces of the treated patients.

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Legends

Fig. 1. A. Before treatment. B. The treatment with the injectable medical devices of the
HAAM was associated with favorable results at a photographic level. C. Before treatment. D.
After treatment Evident reduction of periorbital wrinkles, by improvement of the skin texture,
increase in skin glow and turgidity, which make the skin appear healthier and aesthetically
more beautiful.

Fig. 2. Ultrasound images of the angle of mandibular region A before B after.

Fig. 3. Shows the results of the histological analysis performed on skin biopsy. A. before a
less replicative activity of the epidermis. Van Gieson staining X30. B. After evident
replicative activity of the epidermis. Hematoxylin and Eosin staining X30.

Fig. 4. Histological aspect of epidermis and dermis after the treatment. A. The immuno-
histochemical reaction to the Epidermal Growth Factor (EGF) is quite evident in the stromal
cells and in some epithelial cells. EGF immunohistochemical staining X30. B. More
fibroblasts and collagen fibres were present. Van Gieson staining X30

Hematoxylin and Eosin staining X30

Fig. 5 The bar graphs show the number of pH-metry, sebometry, tissue hydratation, EGF+,
vessels, dermal and epithelium thickness before and after the treatment (Mann-Whitney test).

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Accepted Article Dermal Thickness Epidermal
Sebometry Hydration
thickness pH-metry Epithelium Growth Vessels
[µg/cm2/min] [g/m2/h]
[mm] [µm] Factor (EGF)+

Groups Before After Before After before after before after before after before after before after

Average 1.1 1.41 4.75 5.35 41.5 59.6 55.5 66.6 68.21 140.01 10.5 220.0 4.0 7.75

SD ±0.4 ±0.3 ±0.77 ±1.77 ±2.7 ±3.33 ±2.7 ±3.33 ±1.4 ±0.4 ±2.22 ±11.77 ±1.4 ±1.5

p- value p<0.05 p<0.05 p<0.01 p<0.01 p<0.01 p<0.01 p<0.01

Tab. 1 Summary of the effectiveness before and after the biomodulation treatment (mean,
standard deviation).

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