Research Article
Skin Pharmacology
and Physiology
Skin Pharmacol Physiol 2023;36:140–148
DOI: 10.1159/000529630
Development of an in vitro Functional
Assay to Evaluate the Occlusive
Properties of Moisturizers on Dry Skin
Soonjin Hong Prithwiraj Maitra Audrey Nguyen Kuniko Kadoya
Rahul C. Mehta
Skincare RandD, Allergan Aesthetics, an AbbVie Co., Irvine, CA, USA
Keywords
Dry skin · Moisturizers · Three-dimensional skin model · In vitro
models · Bioassay
Abstract
Introduction: Dry skin is a hallmark of impaired skin
barrier function. Moisturizers are a mainstay of treat-
ment to help the skin retain moisture, and there is a high
consumer demand for effective products. However, the
development and optimization of new formulations are
hampered due to lack of reliable efficacy measures using
in vitro models. Methods: In this study, a microscopy-
based barrier functional assay was developed using an
in vitro skin model of chemically induced barrier damage
to evaluate the occlusive activity of moisturizers.
Results: The assay was validated by demonstrating the
different effects on barrier function between humectant
(glycerol) and occlusive (petrolatum). Significant
changes in barrier function were observed upon tissue
disruption, which was ameliorated by commercial
moisturizing products. Conclusion: This newly devel-
oped experimental method may be helpful to develop
new and improved occlusive moisturizers for the
treatment of dry skin conditions.
© 2023 S. Karger AG, Basel
karger@karger.com © 2023 S. Karger AG, Basel
www.karger.com/spp
Received: January 5, 2022
Accepted: February 1, 2023
Published online: March 2, 2023
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Introduction
Maintaining proper skin hydration is essential for
healthy, flawless-looking skin. Skin hydration is not only
of cosmetic importance but also necessary for the skin to
fulfill “la raison d’être” as described by Madison: acting as
the body’s primary permeability barrier [1]. The outer-
most layer, the stratum corneum (SC), consists of a
network of tightly packed corneocytes surrounded by
intracellular lipids and hygroscopic materials that restrict
the passage of water [2–6]. The permeability of SC is
significantly influenced by the lamellae lipid components
and their structural organization [7–9]. In normal skin, a
relatively low level of water is lost to the environment,
which is constantly replenished by the underlying viable
tissue. This homeostatic level of moisture is vital to
preserve the integrity and smooth appearance of the SC,
as insufficient hydration can compromise its barrier
function.
Xerosis, or dry skin, is a common problem that results
from damage to the barrier and excessive water loss. The
underlying causes of xerosis are diverse and complex and
can result from several individual or environmental
factors. Moisturizers play a central role in a basic skin-
care regimen, and a myriad of products are available
claiming to address dry skin. Active moisturizing
Correspondence to:
Rahul C. Mehta, Rahul.mehta @ abbvie.com
ingredients may work by acting as occlusives, which coat
the skin surface as a hydrophobic film, humectants, which
increase SC hydration by attracting and binding to water,
or emollients that can fill gaps between corneocytes and
smooth the skin [10]. In addition, some products include
actives that stimulate endogenous barrier repair. Mois-
turizers are formulated to fulfill one or several of these
functions, but in general, they aim to prevent further
evaporation (occlusives) or allow water to replenish the
SC from within the deeper layers of the skin (humec-
tants). This increase in water content can both improve
skin appearance and provide sufficient hydration for the
restoration of barrier function.
Despite the wide range of products and formulations
available, effective moisturizers represent an area of high
consumer need [11]. However, the lack of reliable mea-
surements of barrier function using in vitro models poses a
challenge for the preclinical development of new products.
There is a paucity of clinical studies evaluating the efficacy of
moisturizers and even fewer describing their development
and optimization. Comparative assessment of trans-
epidermal water loss (TEWL) is the most common
method used to assess the effect of moisturizers on the skin,
under the premise that a compromised barrier is more
permeable to water. Several different devices exist to
measure TEWL; these have found widespread clinical use
because they are noninvasive. However, because these
measurements rely on measuring vapor density, they are
prone to interference from the environment and require
very careful, controlled conditions [12, 13]. Moreover, these
measurements may not be able to detect more minor
changes in barrier integrity that produce clinically signifi-
cant results [14]. Advanced spectral and imaging techniques
have been recently introduced to measure skin hydration.
While they can provide a vast amount of information re-
garding the structure and composition of the skin, the
instrumentation is expensive, and complex data deconvo-
lution and analysis techniques are required [13]. There is a
need for simple, robust, and higher throughput methods
that can be used to test the effect of moisturizers on skin
barrier function. Thus, the purpose of the study was to
establish a functional assay to measure occlusive properties
of moisturizers in a 3D reconstructed human epidermis
(RHE) model.
Materials and Methods
Materials
EZ-LinkTM NHS-LC-Biotin (N-hydroxysulfosuccinimide [NHS],
cat#21335) was obtained from Thermo Fisher (Waltham, MA, USA),
In vitro Assay Evaluating Moisturizers on
Dry Skin
and 5 mg/mL solution (tracer solution) was prepared in PBS. Lucifer
Yellow (LY) CH dilithium salt (cat#L0259) was purchased from
Sigma (St. Louis, MO, USA) and reconstituted to 1mM in water.
Dimethylpolysiloxane, hyaluronic acid (HA), poly-L-glutamic acid
(PGA) were obtained from Sigma (cat#DMPS5C, cat#53163, and
cat#P4886, respectively). HA and PGA were solubilized to 2% w/v
and 4% w/v in water, respectively. Glycerol was purchased from VWR
(cat#0854, Randor, PA, USA), and petroleum jelly (petrolatum,
Vaseline) was sourced from Unilever (Englewood Cliffs, NJ, USA).
Ultra Sheer Moisturizer (USM) and HA5 Rejuvenating Hydrator
(HA5) that contain both HA and dimethylpolysiloxane were obtained
from SkinMedica (Allergan Aesthetics, an AbbVie Company, Irvine,
CA, USA). The ingredients for USM are as follows: water, cetyl
ethylhexanoate, sorbitan stearate, methyl gluceth-20, polysorbate 60,
dimethicone, tocopherol, tocopheryl acetate, tetrahexyldecyl ascor-
bate, panthenol, sodium hyaluronate, cetearyl alcohol, Ceteareth-20,
aminomethyl propanol, carbomer, disodium EDTA, phenoxyethanol,
ethylhexylglycerin, potassium sorbate. The ingredients for HA5 are as
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follows: water, dimethicone, HDI/trimethylol hexyllactone cross-
polymer, glycerin, butylene glycol, polysilicone-11, Bis-PEG-8
dimethicone, sodium acrylate/sodium acryloyldimethyl taurate co-
polymer, sodium hyaluronate crosspolymer, hydrolyzed HA, sodium
hyaluronate, Vitis vinifera (grape) flower cell extract, Vibrio algino-
lyticus ferment filtrate, Alteromonas ferment extract, Porphyridium
cruentum extract, whey protein, plankton extract, trehalose, urea,
serine, algin, caprylyl glycol, pullulan, disodium phosphate, potassium
phosphate, pentylene glycol, polymethylsilsesquioxane, glyceryl pol-
yacrylate, sodium citrate, sea water, sucrose palmitate, tocopheryl
acetate, hydroxyacetophenone, polysorbate 60, propanediol, potas-
sium sorbate, citric acid, isohexadecane, polysorbate 80, silica, decyl
glucoside, tromethamine, ethylhexylglycerin, phenoxyethanol,
disodium EDTA.
Normal and Dry-Skin Mimicking 3D Human Skin Model
Epiderm™ (3D epidermis human skin model) and Epi-
dermFT™ (3D full thickness human skin model) tissues were
obtained and maintained in culture according to the manufac-
turer’s recommendation (MatTek Corp., Ashland, MA, USA).
Briefly, after being equilibrated overnight, tissues were first treated
topically with either water (control) or 2% sodium dodecyl sulfate
(SDS) for 2 h in a 37°C/5% CO2 incubator. Next, 10 μL of water,
single active ingredients, or commercial moisturizing products
were applied to the surface for an additional 1 h incubation before
being subjected to the tracer assay.
Tracer Assay (Surface Biotinylation Assay)
After topical treatment, tissues were placed in the tracer
solution for 4 min at room temperature while blowing air (4 L/
min) on top of the tissue to induce fast water evaporation.
Tissues were then fixed with 10% neutral buffered formalin,
embedded into paraffin blocks, and processed for immuno-
histochemistry. Tissues were sectioned (4 µm), mounted,
stained with streptavidin-HRP, and tracer migration was vi-
sualized with 3,3’-diaminobenzidine (cat#k3468, Agilent
Technologies, Santa Clara, CA, USA). Images of whole tissue
sections were captured by the NanoZoomer digital slide scanner
(model C9600-02, Hamamatsu Photonics, Hamamatsu, Japan).
The uptake height of the 3,3’-diaminobenzidine signal and the
distance from the bottom of the tissue to the stratum
Skin Pharmacol Physiol 2023;36:140–148 141
DOI: 10.1159/000529630
granulosum (SG) were measured using NDP view2 software
(Hamamatsu) and expressed as the relative distance traveled
using the following equation:
X evaporation apparatus was designed to blow compressed
Relative distance (%)  100
Y*
where X = height of tracer and Y = height of SG.
The relative distance per biological replicate was expressed as
an average of 5 randomly chosen points per tissue.
LY Barrier Assay
Twenty microliters of an LY solution (1 mM) was placed on the
top of EpidermFT™ tissues treated as described above. After
incubation for 1 h, tissues were fixed with 10% neutral buffered
formalin, embedded into paraffin blocks, sectioned (4 µm), and
then mounted on a glass slide. Fluorescent and phase-contrast
images were taken with a REVOLVE microscope equipped with
×10 objective (Echo, San Diego, CA, USA).
Confocal Raman Microspectroscopy
Water content in the skin was measured using the gen2-SCA
performance confocal Raman system (RiverD International,
Rotterdam, The Netherlands) [15, 16]. Raman spectra in the high
wave number region (2,500–4,000 cm-1) were acquired with
×60 oil-immersion objective using an excitation wavelength of 671
nm and 2 s exposure time. Measurements were taken in incre-
ments of 2 μm from the skin’s surface down to a depth of 70 μm.
The average of 4 readings was obtained from each sample, which
was repeated across 3 different skin samples. Raman spectra were
analyzed using the embedded software (SkinTools 3, RiverD).
Statistical Analysis
Data analysis was conducted in R, and figures were produced
using the package ggplot2 [17].
Results
Tracer Assay (Surface Biotinylation) Visualizes the
Water Loss from the Top Layer of RHE
To quantify the occlusive ability of moisturizers, an
“inside-out” tracer assay was adapted from the surface
biotinylation method to indirectly measure the migration
of water through 3D organotypic skin culture [18]. The
tracer molecule (NHS-Biotin) is dissolved in PBS that is
in contact with the basal keratinocyte layer, where it can
then migrate upward due to the moisture evaporation
from the top surface (Fig. 1a). Since the NHS-LC-biotin
tracer is cell impermeable, it may also react with proteins
containing N-terminal amino groups and exposed lysine
residues during its migration through the paracellular
track. They can be structural transmembrane proteins
that constitute adherens junctions (E-cadherin), des-
mosomes (desmoglein), tight junctions (claudin), and
extracellularly secreted proteins, such as perlecan. Al-
though tight junctions halt the movement of NHS-Biotin
142 Skin Pharmacol Physiol 2023;36:140–148
DOI: 10.1159/000529630
at the SG, a shift in the water gradient will affect how fast
the tracer moves paracellularly. To demonstrate the link
between water loss and tracer migration, a forced
air at the rate of 4 L/min directly on the top center surface
of RHE (Fig. 1a). Differences in permeability were cal-
culated as the relative distance traveled by the tracer
(Fig. 1b, bottom). Natural evaporation did not induce a
noticeable tracer movement (Fig. 1b, c). In contrast,
forced evaporation significantly induced tracer migration
toward the SC-SG layer, indicating that topical water loss
promoted tracer migration (Fig. 1b, c). Considering that
the migration in dry skin would be higher, and topical
occlusion would reduce migration, a 4 min experimental
duration was chosen to evaluate dry skin barrier function
and improvement (Fig. 1d).
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Measuring Tracer Flux in a 3D Human Skin Model
Can Show Compromised Skin Barrier Function
To mimic dry skin, the skin barrier function was
acutely compromised by applying a strong anionic de-
tergent SDS to the SC at the air-liquid interface [19]. The
SDS-treated tissue displayed an increased distance trav-
eled by the tracer media compared to control-treated
tissue (Fig. 2a, b). Thus, tissue barrier function can be
comparatively measured by the tracer flux in this system.
Independent experiments from the “outside-in” LY assay
and confocal Raman microspectroscopy were performed
to support the observation that SDS treatment disrupts
SC barrier integrity (Fig. 2c, d) [19–21]. With LY, a
deeper and brighter fluorescent signal in SDS-treated
tissue was observed compared to control, representing
the compromised outer barrier (Fig. 2c). Confocal Raman
microspectroscopy was then used to measure the water
content in the SC and its below layers, as previously
described in the literature [16, 22]. Both control- and
SDS-treated skin were exposed to forced evaporation,
without contact with PBS. Whereas normal skin main-
tained 70% of water content (Iwater/Iprotein) below ~30 µm
skin depth, in SDS-treated skin it dropped to between 50
and 60% (Fig. 2d).
If the movement of the tracer is reflective of water loss,
then the passage of the tracer should be influenced by the
properties of topically applied material. To validate the
assay, both a humectant (glycerol) and an occlusive
(petrolatum) were tested on control- and SDS-treated
skin. In normal skin, the petrolatum reduced the relative
distance of tracer uptake, while that of glycerol was
greater than the water-treated condition (Fig. 3a, b). As
expected, SDS treatment induced higher tracer migration,
whereas topical application of petrolatum reduced
Hong/Maitra/Nguyen/Kadoya/Mehta
 a
c d
Fig. 1. Surface biotinylation assay visualizes the water loss from top
layer of skin, which represents skin barrier function. a A schematic
depicting the well containing the tissue, tracer, and air-liquid
interface for topical administration (left, figure created on
Biorender.com). The forced evaporation system constructed for
the EpiDerm™ tissue culture format (right). b Topical moisture
evaporation naturally (left) or induced by blowing air (4L/min) for
10 min of linker treatment, processed for immunohistochemistry
with streptavidin-HRP and DAB. The uptake height of the tracer
migration in both control- and SDS-treated tissue. In
contrast, the humectant only provided marginal im-
provement to normal skin tissue barrier integrity
(Fig. 3a, c).
Both Single Ingredients and Commercial Products
Modulate Skin Barrier Function
To demonstrate the utility of the tracer assay, the
occlusive capabilities of either single ingredients or
comprehensive products were tested. An occlusive,
dimethicone, as well as 2 humectants HA and PGA were
In vitro Assay Evaluating Moisturizers on
Dry Skin
b
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(X) was expressed as a relative distance traveled toward the SG (Y).
This was quantified from 5 distinct points per tissue. c Quanti-
fication of the conditions in (b). d With the forced evaporation,
EpiDerm™ tissues were treated for different and the relative
distances were calculated as described in (c). Significant differences
were determined using Students’ T test; **p ≤ 0.01, ***p ≤ 0.001,
****p ≤ 0.0001, n.s. indicates not significant. Data are shown as
mean ± standard deviation; N = 3 tissues per condition. DAB, 3,3’-
diaminobenzidine.
tested on control- and SDS-treated skin as single in-
gredients (Fig. 4). For comparison, 2 commercial cos-
metic moisturizers USM and HA5 that contain one or
more of the above ingredients were also tested using this
platform. As expected, dimethicone reduced the relative
distance in both control- and SDS-treated skin. However,
while both 2% HA and 4% PGA increased the tracer
migration in control skin, PGA additionally decreased
that in SDS-treated skin. Despite these differences, both
USM and HA5 reduced tracer migration on control- and
SDS-treated skin.
Skin Pharmacol Physiol 2023;36:140–148 143
DOI: 10.1159/000529630
 a
c
d
Fig. 2. Measuring tracer flux in a 3D human skin model is indicative
of compromised skin barrier function. a Control tissue (left) or
tissue treated with SDS (2%) after 4 min of linker treatment,
processed for immunohistochemistry with streptavidin-HRP and
DAB. b Quantification of the conditions in (a). Significant differ-
ences were determined using Students’ T test; ****p ≤ 0.0001. Data
shown as mean ± standard deviation; n = 3 tissues per condition.
Discussion
Having a robust in vitro platform can facilitate the
optimization of moisturizer formulations. The ap-
proach described here represents a simple and acces-
sible methodology to evaluate the physical effects of
moisturizing products in an in vitro model of dry skin.
SDS, a severe irritant previously established to interfere
144 Skin Pharmacol Physiol 2023;36:140–148
DOI: 10.1159/000529630
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c Tissues were treated with either control (water) or SDS (2%), then
LY to measure the skin barrier integrity. d Confocal Raman
spectroscopic measurements were performed for water content in
EpiDerm™ tissues treated either with water or SDS (2%) under
forced evaporation. Water contents were depicted along the skin
depth (~70 µm). Mean value was represented by dotted line and
standard error by shades. DAB, 3,3’-diaminobenzidine.
with the SC, was used to disrupt skin barrier integrity
[19, 23], and the degree of damage was measured by the
migration of an aqueous small molecule tracer. It was
shown that the application of an occlusive moisturizing
product was able to reduce the uptake of the tracer and
thus water loss (Fig. 3, 4). Ideal moisturizers not only
physically prevent the water loss from the top surface
but also replenish water molecules from deep within the
Hong/Maitra/Nguyen/Kadoya/Mehta

a b
Fig. 3. Topical applications of humectants and occlusives
modulate skin barrier function. a The tracer assay outlined in
Fig. 2 was performed, using SDS treatment followed by the
additional topical application of water, glycerol (humectant), or
petrolatum (occlusive). b Relative distance of the conditions in
(a), comparing the effects of glycerol or petrolatum, in either
control- or SDS-treated skin. Significant differences were de-
termined using Students’ T test; ****p ≤ 0.0001, n.s. indicates
skin to maintain the physiological condition up to the
SC. For this reason, most moisturizers contain a
mixture of ingredients of two opposite properties
– occlusives and humectants, but fine-tuning to max-
imize each property is a difficult task. While dime-
thicone hindered the tracer migration in both normal
and dry skin models, the humectants HA and PGA
increased tracer migration in the control skin condition
(Fig. 4). It is reasonable to postulate that topical water
evaporation drew the tracer solution by capillary action
and water-holding humectant properties facilitate its
In vitro Assay Evaluating Moisturizers on
Dry Skin
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not significant. c Relative distance of the conditions in (a),
comparing tracer migration in control- and SDS-treated skin in
the presence of glycerol or petrolatum. Significant differences
were determined using Students’ T test; **p ≤ 0.01, ****p ≤
0.0001, n.s. indicates not significant. In figure b, c, the relative
distance was calculated from 5 different points per tissue. For all
figures, data are shown as mean ± standard deviation. N = 3
tissues per condition.
continuity. However, it is not clear why in the SDS-
treated model higher tracer uptake was not observed.
One possible explanation is that the disruption of SC
enlarged the paracellular space in SC and interfered
with capillary action. The opposite effects from hu-
mectants and occlusives and their dosage level deter-
mine overall efficacy of the final formulation as shown
in the tracer migration of USM and HA5, although
other ingredients likely play a role. Individual ingre-
dient assessment using this tracer assay could give
insights on how to balance these two properties.
Skin Pharmacol Physiol 2023;36:140–148 145
DOI: 10.1159/000529630
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Fig. 4. Degree of skin barrier function varies upon topical applications of single ingredient and commercial
products. The tracer assay outlined in Fig. 2 was performed, using SDS (2%) treatment, followed by the additional
topical application of silicone, HA, polyglutamic acid, or commercial moisturizer/hydrator (USM or HA5). The
non-treated average levels of water and SDS-treated skin were noted by red and orange dotted lines. Data are
shown as mean ± standard deviation. N = 3 tissues per condition.

This system has several advantages over previously
used methodologies to evaluate barrier integrity [24].
First, the advent of RHE removes the ethical constraints
of in vivo experiments and the tedious recruitment of
human volunteers. The test system can thus be cus-
tomized to include other methods of barrier disruption,
and the use of RHE from donors of different skin types
and ethnicities can address some of the biological vari-
ables that would affect future clinical results [25]. Second,
while the classic, gold standard measurements such as
TEWL and skin capacitance have utility in the clinic
because they are noninvasive, they are prone to inter-
ference and require careful standardization between
conditions [12]. This system uses reliable cellular and
molecular biology techniques and controlled laboratory
conditions, removing many of the variables associated
with experimental error. In this tracer assay, the imaging
measurements and calculations are simple and
straightforward. Therefore, many samples can be pro-
cessed in parallel, making this approach highly amenable
to structure-activity relationship studies and rapid
screening of different formulations.
While this methodology was developed for cosmetic
dry skin, it can be extended to other in vitro models of
conditions that disrupt barrier function. In addition to
what was shown here, the tracer assay can be combined
with other endpoints for a more detailed analysis of
changes to the skin. For example, biophysical, analytical,
and atomic simulation techniques used to evaluate the
structure, morphology, and molecular composition
would complement the measurements of barrier integrity
by the tracer assay described herein [26–30]. Especially
since SDS treatment was reported to disrupt long peri-
odicity phase of lipid assembly significantly, rather than
short periodicity phase, it would be interesting to dem-
onstrate the functional roles of long periodicity phase and
short periodicity phase in skin permeability [7, 8, 23]. If
there are reagents to specifically alter the nanostructure of
the lipid matrix in SC, it can be explored how the lipid
matrix contributes to overall SC barrier function with the
tracer assay [31]. Altogether, these complementary assays
can be excellent tools to further optimize the properties of
moisturizers for cosmetic dry skin and possibly other
indications.
There are some limitations to the methodology as
presented here, namely, due to fundamental differences
between 3D in vitro models as compared to native skin.
Cultured 3D skin may be more permeable compared to
human epidermis due to differences in lipid composition
and organization, but consequences of this may be
mitigated because the results from this assay are com-
parative [32]. The possible differences can be addressed in
the future by using additional models, such as ex vivo skin
explants or cultured biopsies [33]. Importantly, proper
management of dry skin often requires repeated, long-
term application of a product. Some formulations can
improve barrier function in the short term, but they may
cause long-term damage when used repeatedly [34]. In

146 Skin Pharmacol Physiol 2023;36:140–148 Hong/Maitra/Nguyen/Kadoya/Mehta

DOI: 10.1159/000529630
this study, the effect of a single application was examined Conflict of Interest Statement
but could potentially be extended up to several days.
Because the tracer assay cannot be employed in patients, Soonjin Hong, Prithwiraj Maitra, Audrey Nguyen, Kuniko
Kadoya, and Rahul Mehta are employees of AbbVie, Inc., and may
it is currently unclear if the results from this assay would own stock in the company.
be predictive of efficacy in vivo. In the future, it will be
important to relate the results from the barrier assay
in vitro to those noninvasively obtained in comple- Funding Sources
mentary clinical studies.
This research was supported by Allergan Aesthetics, an AbbVie
company. Employees of AbbVie participated in the research,
Conclusion interpretation of data, review of the manuscript, and the decision
to submit for publication.
In summary, a simple and practical method was de-
veloped to assess moisturizing ingredients and formu-
Author Contributions
lations in terms of occlusive effects in vitro. This
approach may be a valuable tool for the preclinical de-

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velopment and optimization of new moisturizing for-
mulations to treat cosmetic dry skin.
Acknowledgment
Writing assistance and editorial support were provided by Liza
Selwan-Lewis, PhD, an employee of AbbVie, Inc.
Soonjin Hong, Prithwiraj Maitra, Kuniko Kadoya, and Rahul
Mehta were responsible for the conception and design of the work.
Soonjin Hong was responsible for the acquisition, analysis, and in-
terpretation of the results. Audrey Nguyen was responsible for the
histological sample preparation. All authors provided critical feedback
for the manuscript as developed, reviewed and approved the man-
uscript, and have agreed to be accountable for all aspects of the work
in ensuring that questions related to the accuracy or integrity of any
part of the work are appropriately investigated and resolved.

Data Availability Statement

Statement of Ethics
All data generated or analyzed during this study are included in
Ethical approval was not required for this study type, and no this article. Further inquiries can be directed to the corresponding
human or animal subjects or materials were used. author.

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