Article

Evaluation of the Effectiveness of an Innovative

Polycomponent Formulation on Adult and Aged Human
Dermal Fibroblasts
Francesca Rosaria Augello 1,†, Francesca Lombardi 1,†, Serena Artone 2, Alessia Ciafarone 3, Serena Altamura 2,
Luisa Di Marzio 4, Maria Grazia Cifone 1, Paola Palumbo 1, Maurizio Giuliani 1,5 and Benedetta Cinque 1,*

1 Department of Life, Health & Environmental Sciences, University of L’Aquila, 67100 L’Aquila, Italy;
francescarosaria.augello@univaq.it (F.R.A.); maurizio.giuliani@univaq.it (M.G.)
2 PhD School in Medicine and Public Health, Department of Life, Health and Environmental Sciences,

University of L’Aquila, 67100 L’Aquila, Italy

3 PhD School in Health & Environmental Sciences, Department of Life, Health and Environmental Sciences,

University of L’Aquila, 67100 L’Aquila, Italy

4 Department of Pharmacy, University of Chieti—Pescara “G. D’Annunzio”, 66100 Chieti, Italy

5 Unit of Plastic and Reconstructive Surgery, Casa Di Cura Di Lorenzo SrL, Via Vittorio Veneto 37,

67051 Avezzano, Italy

* Correspondence: benedetta.cinque@univaq.it; Tel.: +39-0862-433-553
† These authors contributed equally to this work.

Abstract: Skin aging is a dynamic process that determines structural alterations in ECM and
reduction in dermal fibroblasts. The recent availability on the market of an innovative
polycomponent formulation (KARISMA Rh Collagen® FACE, K) containing noncrosslinked high-
molecular-weight hyaluronic acid (HMW-HA), a human recombinant polypeptide of collagen-1
alpha chain, and carboxymethyl cellulose (CMC), attracted our scientific interest in evaluating its
biomolecular effects on human dermal adult and aged fibroblasts. After treatment with increasing
Citation: Augello, F.R.; Lombardi, F.; K concentrations, cell proliferation, collagen I, prolyl 4-hydroxylase (P4HA1), an essential protein
Artone, S.; Ciafarone, A.; Altamura, S.; in collagen biosynthesis, and α-SMA levels were assessed. The fibroblast contractility, TGF-β1
Di Marzio, L.; Cifone, M.G.;
levels, and oxidative stress markers were also evaluated. K formulation exposure led to a significant
Palumbo, P.; Giuliani, M.; Cinque, B.
and dose-dependent increase in the proliferation and migration of adult fibroblasts. Of note, the K
Evaluation of the Effectiveness of an
exposure counteracted the H2O2-induced aging by promoting cell proliferation, reducing β-
Innovative Polycomponent
galactosidase activity, and neutralizing the aging-associated oxidative damage. Moreover, an
Formulation on Adult and Aged
Human Dermal Fibroblasts.
increase in collagen I, P4HA1, α-SMA, TGF-β1 levels, and improved contractility of adult and aged
Biomedicines 2023, 11, 2410. fibroblasts were observed after treatment. Overall, our results show evidence that the K treatment
https://doi.org/10.3390/biomedicines is efficacious in improving biological functions in adult fibroblasts and suppressing the
11092410 biomolecular events associated with H2O2-induced cellular aging, thus supporting the regenerative
and bio-revitalizing action of the K formulation helpful in preventing or treating skin aging.
Academic Editor: Shaker A. Mousa

Received: 3 August 2023 Keywords: polycomponent formulation; skin aging; dermal fibroblast; human recombinant
Revised: 24 August 2023 polypeptide of collagen-1 α chain; TGF-β1; oxidative stress
Accepted: 26 August 2023
Published: 28 August 2023

1. Introduction
Copyright: © 2023 by the authors.
Skin structure and physiology are continuously subjected to intrinsic and extrinsic
Submitted for possible open-access
factors which gradually damage the skin homeostasis, inducing a progressive loss of
publication under the terms and
tissue integrity and impairment of cellular functions. At the tissue level, the epidermis
conditions of the Creative Commons
Attribution (CC BY) license
becomes thinner due to a progressive slowing of keratinocyte proliferation and
(https://creativecommons.org/license
differentiation failing. Of note, the stimulation of the regenerative capacity of
s/by/4.0/). keratinocytes is a potential target in antiaging therapy [1,2]. Regarding the dermal
compartment, the cellular and molecular mechanisms underlying skin aging are

Biomedicines 2023, 11, 2410. https://doi.org/10.3390/biomedicines11092410 www.mdpi.com/journal/biomedicines

Biomedicines 2023, 11, 2410 2 of 16

associated with the dermal fibroblasts in terms of number and functionality and with the
alteration of ECM homeostasis, involving mainly collagen, the crucial fiber-forming
structural element. [3]. In the aged skin, the number of dermal fibroblasts functionally
active and responsible for the synthesis of the ECM elements is reduced while an
accumulation of aged fibroblasts occurs. These cells are characterized by altered metabolic
functions with reduced collagen synthesis in favor of increased secretion of inflammatory
cytokines, chemokines, and matrix metalloproteinases (MMPs), degradative enzymes of
ECM components, such as collagen fibrils [4]. All these characteristic features significantly
reduce the skin’s regenerative potential [5]. In the process of aging, the skin structure and
integrity are impaired with changes in mechanical properties and viscoelasticity due to
the exhaustion of the ECM components, such as collagen, elastin, and hyaluronic acid
(HA) [6]. Then, during the aging process, the ECM, which generally serves as the physical
support structure for the dermal connective tissue and makes up the appropriate
environment for active and functional dermal cells, is altered and impacts the functions
of the fibroblasts that cannot adhere to it, being the mechanical forces reduced. The aged
dermal fibroblasts appear with collapsed cytoplasm, rounded shape, and, in particular, a
reduced capacity of type I collagen production [7].
For the above, dermal fibroblasts represent one of the most exciting targets of skin
aging prevention or therapy.
To hinder or counteract the critical signs of skin aging, the injectable hydrogel is one
of the most used approaches due to peculiar physicochemical characteristics. The known
clinical efficacy attracted our scientific interest in evaluating deeper the biological effects
on in vitro cellular models. This innovative commercially available polycomponent
formulation (KARISMA Rh Collagen® FACE, K) includes three components, generally
used individually. In particular, K formulation contains, in well-defined proportions,
noncrosslinked high-molecular-weight HA (HMW-HA), recombinant polypeptide of
collagen-1 chain, and carboxymethyl cellulose (CMC). HA is an essential constituent of
the extracellular matrix with multiple biological activities, including hydration, that
contribute to the firmness and bounciness of the skin. Its hydrophilic properties draw
water, endowing improved viscoelasticity to the skin. The literature data extensively
report that the hydrogels containing HA are able to improve skin appearance and texture
[8,9]. Clinical observations demonstrate that the positive effects of injectable hydrogels
can be persistent, going beyond the filling impact [10], suggesting that other mechanisms
could be involved to stimulate ex novo collagen synthesis and generally improve the
functionality of fibroblasts. The innovative polycomponent K formulation includes the
recombinant polypeptide α1 chain of type I human collagen synthesized by transgenic
silkworms. Due to its chemical structure, the recombinant α1 chain cannot form the
collagen triple helix [11]. When used at concentrations > 5 µg/mL, it promoted the
adhesivity and spread of human dermal fibroblasts (HDFs) similarly to gelatin or native
collagen. The results suggested the advantageous use of recombinant α1 chain of collagen
as biomaterial for a variety of medical applications, having a low risk of bacterial
contaminations and a quality of product easily controlled [11]. It is important to highlight
that the collagen for injectable formulations is generally extracted from different animal
sources [12,13], and that the human collagen formulations are also available as a valid
alternative. However, some pitfalls can be found, such as long preparation times, high
costs for the procedure, and highly skilled teams, so an exciting alternative is represented
by recombinant collagens [14]. Carboxymethylcellulose (CMC) is an FDA approved
water-soluble polysaccharide, derived from cellulose [15]. Of note, CMC tends to disperse
in water to form a transparent colloidal solution, i.e., CMC gel, and it is also able to
suspend solids in aqueous media and stabilize emulsions. It is also used as an agent to
thicken, bind, and gel and to retain water [16]. The CMC has been used in the biomedical
field due to its biocompatibility, low cost, nontoxicity, good biodegradability, and low
immunogenicity. The numerous rheological properties of CMC such as mechanical
strength, different formability, tunable hydrophilicity, and viscosity justify a wide range
Biomedicines 2023, 11, 2410 3 of 16

of applications [17] rendering the CMC the material of choice in injectable formulations
[15,18]. Among CMC-based hybrids, the hydrogel has gained much interest also in
pharmaceutical and cosmetic applications, since the CMC ensures the recommended
rheological properties due to its viscosity. In soft tissue engineering, the CMC hydrogels
offer a softer feel via high spreading accessibility improving the dermal formulation
administration and making it less viscous [19].
Herein, the effects of K formulation treatment at different concentrations and for
different incubation times were evaluated on proliferation, migration, and contraction of
adult and aged HDFs. The influence of K formulation exposure on type I collagen
synthesis pathway was also assessed. Considering the key role of TGF-β1 pathway on
collagen I synthesis, the modulation of its levels was also investigated in both adult and
aged dermal fibroblasts after treatment with K formulation. Furthermore, the ability of K
formulation to counteract oxidative stress associated with H2O2-induced aging process in
fibroblasts was verified. Our findings support the efficacy of K formulation as a promising
strategy for maintaining dermal fibroblast homeostasis and inhibiting critical age-related
skin signs.

2. Materials and Methods

2.1. Preparation of K Polycomponent Formulation for Cell Treatments
KARISMA Rh Collagen® FACE, a bio-restorative formulation (following named K)
(kindly provided by Taumedika Srl, Rome, Italy), includes 200 mg/mL high-molecular-
weight hyaluronic acid (HA), 200 µg/mL human recombinant collagen α1 chain, and 40
mg/mL carboxymethylcellulose (CMC). For cell treatments, K was prepared using the
“extraction dilution method” as described by the UNI EN ISO 10993 regulation [20].
Briefly, the procedure was carried out in an extraction medium consisting of DMEM,
supplemented with 10% of fetal bovine serum (FBS), 100 U/mL penicillin, 100 mg/mL
streptomycin, and 2 mM glutamine (complete medium) at 37 °C ± 1 for 24 h, by continuous
agitation. Subsequently, K was serially diluted and used at different concentrations
(range: 0.1–10% v/v, final concentration).

2.2. Cell Systems

Adult normal primary human dermal fibroblast (NHDFs, Adult CC-2511) cell line,
derived from a 47-year-old Caucasian female, was obtained from Lonza/Cell Applications
(Basel, Switzerland) and cultured at low passage (N = 6–9) in complete medium in a
humidified atmosphere of 95% air and 5% CO2 at 37 °C. All reagents and consumables
were purchased from EuroClone (West York, UK). At the 15th passage the cells were used
for the experiments.
To induce the aged phenotype, NHDFs at 70% confluence were treated with 25 µM
hydrogen peroxide (H2O2) in PBS for 1 h. Then, the H2O2 solution was replaced with the
complete medium. After 24 h, the cells (aged HDFs) were treated with K formulation at
the different concentrations.

2.3. Treatments and Assessment of Cell Viability and Growth Rate

Cell growth rate was measured by IncuCyte® Live Cell Imager system (Essen
BioSciences, Inc., Ann Arbor, MI, USA) for real-time analysis of cell confluence. Briefly,
adult or aged HDFs were seeded on a 96-well culture plate at 2.5 × 103 cells/well and
allowed to attach overnight. After, the cells were incubated with increasing concentrations
(0.1, 0.5, 2, 5, or 10% vol/vol, final concentration) of K. Each treatment condition was
replicated in three different wells, and three independent experiments were carried out.
Culture plates were placed into IncuCyte® instrument, and images were acquired every 4
h from 0 to 72 h after treatment. Two pictures were obtained from several points of the
well, at 10× magnification. To measure the proliferation rate, cell confluence was analyzed
by IncuCyte ZOOM™ software (2020b, Essen Bioscience, Newark, UK).
Biomedicines 2023, 11, 2410 4 of 16

To analyze their number and viability, adult or aged HDFs were seeded at a density
of 7000 cells/cm2, grown for 18 h, and incubated with K at 0.1–10% final concentration for
72 h in complete medium. Then, the cells were collected, centrifuged for 10 min at 400× g,
and counted with 0.04% trypan blue (EuroClone, West York, UK), using a Bürker
chamber, and visualized with Eclipse 50i microscopy (Nikon, Tokyo, Japan). Untreated cells
were used as controls. For all the other experiments, similar cell conditions were used.

2.4. Staining Cells for β-Galactosidase Activity

β-Galactosidase activity, a indices of dermal fibroblast cellular aging in vitro, was
assayed using a kit by Cell Signaling Technology (Danvers, MA, USA) according to the
manufacturer’s instructions. After treatments, the aged HDFs were washed with cold PBS
and fixed for 15 min with 1 mL of fixing buffer at room temperature. After washing, the cells
were incubated with 1 mL of β-galactosidase staining solution containing 5-bromo-4-chloro-
3-indolyl-β-d-galactopyranoside (X-gal) for 24 h at 37 °C. Images were obtained using a light
microscope (Eclipse 50i, Nikon, Tokyo, Japan). Ten random fields were counted to
determine the percentage of β-galactosidase-positive cells in the total cell population.

2.5. Fluorometric Measurement of Reactive Oxygen Species (ROS) with DCFH-DA

Intracellular ROS production was evaluated using a DCFH-DA probe
(Immunological Sciences, Rome, Italy). The DCFH-DA probe is cell permeable and is
subjected to deacetylation by esterase to produce nonfluorescent DCFH, which is retained
in the cytosol. In the presence of ROS, DCFH is oxidized to fluorochrome 2′,7′-
dichlorofluorescein. Thus, this probe has been utilized as an indicator of oxidative stress
in biological systems. Briefly, after treatments, the aged HDFs were incubated with
DCFH-DA solution (25 µM) at 37 °C for 30 min. ROS levels were then analyzed using
VICTORX4™ fluorometer (PerkinElmer, Waltham, MA, USA) with excitation and
emission filters of 488 and 535 nm, respectively. The values obtained were normalized for
cell number and expressed as relative fluorescence unit (RFU)/105 cells.

2.6. Malondialdehyde (MDA) ELISA Kit

The aged HDFs supernatants, recovered following treatment with K at 0.5, 2, or 5%
up to 72 h, were assayed for malondialdehyde (MDA) levels by an enzyme-linked
immunosorbent assay (ELISA) kit (Elabscience, Houston, TX, USA) as described in the
manufacturer’s instructions. The values obtained were normalized for cell number and
expressed as ng/105 cells.

2.7. Western Blot Analysis

Western blot was used for protein detection. Cell pellets were washed in PBS and
then lysed in RIPA Lysis Buffer (Merck KGaA, Darmstadt, Germany) added with 100 mM
protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Then, to eliminate cell
debris, the samples were centrifuged at 17,949× g, and the total protein concentration was
determined by DC Protein Assay (BioRad, Hercules, CA, USA). A total of 25 µg of proteins
was separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto 0.45
µm nitrocellulose membrane sheets (BioRad) for 1 h at 4 °C. We used 5% nonfat dry milk
to block the aspecific sites on membranes (one hour at room temperature). The
membranes were then incubated overnight at 4 °C with rabbit polyclonal antibody anti-
COL1A1 (Boster Biological Technology, Pleasanton, CA, USA) 1:1000, rabbit monoclonal
antibody anti-P4HA1 (OriGene, Rockville, MA, USA) 1:1000, or mouse monoclonal
antibody anti-α-actin smooth muscle (ACTA2, α-SMA) (OriGene, Rockville, MA, USA)
1:1000 and mouse monoclonal antibody anti-GAPDH (OriGene, Rockville, MA, USA)
1:1000. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit or rabbit anti-mouse
IgG secondary antibodies at 1:2000 were used (Millipore EMD, Darmstadt, Germany). The
densities of immunoreactive bands visualized by chemiluminescence reagent (ECL,
Biomedicines 2023, 11, 2410 5 of 16

Amersham Pharmacia Biotech, Buckinghamshire, UK) were quantified by

chemiluminescence documentation system ALLIANCE (UVITEC, Cambridge UK). The
data were normalized to the relative GAPDH bands.

2.8. Immunofluorescence Assay for Type I Collagen

Adult or aged HDFs were grown on coverslips of a 12-well plate (seeded at 7000
cells/cm2) and untreated or treated with K at 5% for 72 h. At the end of treatment, the cells
on the coverslips, after washing with PBS and formaldehyde fixation (4% for 20 min), were
permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 5 min and
blocked with 3% BSA (Sigma-Aldrich) for 20 min at room temperature. After that,
coverslips were incubated for 18 h at 4 °C with rabbit polyclonal antibody anti-COL1A1
(Boster Biological Technology, Pleasanton, CA, USA) at dilution 1:250. FITC conjugated
goat anti-rabbit polyclonal IgG secondary antibody (Millipore EMD, Darmstadt,
Germany) was used at 1:1000 for 1 h at room temperature. VECTASHIELD® Antifade
Mounting Medium with added DAPI (Vector Laboratories, Inc., Burlingame, CA, USA)
was used to mount the coverslips examined at 100× magnifications by fluorescence
microscopy (Eclipse 50i, Nikon, Tokyo, Japan).

2.9. Extracellular Collagen Quantification

The extracellular collagen from cell culture supernatants was quantified using the
human type I collagen ELISA kit (Immunological Sciences, Rome, Italy). Adult and aged
HDFs were grown on a 12-well plate (seeded at 7000 cells/cm2) and treated with K at 0.5,
2, or 5% for 72 h in a complete medium. Following the K exposure, the media were
recovered and centrifuged at 1000× g for 15 min to remove the cellular debris/dead cells.
The concentration of collagen I was assayed, and the obtained values were normalized for
cell number and expressed as ng/105 cells.

2.10. In Vitro Scratch Assay

The effect of K formulation on migration/proliferation in adult HDFs was carried out
by scratch assay [21]. The cells were seeded at 20 × 104/cm2 on multiwell plates and left to
grow until reaching confluence. In the absence of the medium, the cell monolayers were
scratched with a 200 µL pipet tip and then washed with PBS to remove debris. Fresh
medium, containing or not K at 0.5, 2, or 5%, was added to the scratched monolayers. The
images were acquired by the inverted light microscope (Eclipse TS100, Nikon, Tokyo,
Japan) at different time points after the scratch (0–24 h). For the quantitative analysis, the
standalone TScratch software was used. The software was able to measure the portion of
the area that was occupied by the cells by a mathematical model and then to calculate the
percentage of wound closure. The experiments were performed in duplicate, and six fields
for each condition were evaluated.

2.11. Collagen Gel Retraction Assay

To evaluate the contraction ability of adult or aged HDFs, the fibroblasts were put
into three-dimensional collagen lattices as previously described [22]. Briefly, acid-
extracted type I collagen (5 mg/mL) mixture was made on ice with rat tail collagen I (Enzo
Life Sciences, Lausen, Switzerland) in acetic acid 0.2%. The solution was then diluted at 3
mg/mL in sterile water, and the cells were resuspended in complete media (8 × 105 cells/mL).
Resuspended cells (8 × 104/100 µL) were mixed to 300 µL complete media, 200 µL
collagen mixture (3 mg/mL), and the mixture was quickly neutralized with NaOH on ice.
These treatments did not influence the viability and the number of fibroblasts, as revealed
by Trypan blue exclusion test (not shown). A total of 500 µL of the collagen/cell mixture
was placed into each well of a pre-warmed 12-well plate (Corning Incorporated, Corning,
NY, USA). After gel polymerization (at 37 °C for 1 h), the media containing or not K (0.5, 2, or
5%) were added in each well. Then, the lattices were accurately detached from the well. The
Biomedicines 2023, 11, 2410 6 of 16

images of lattices were taken with a digital camera before release and multiple times after
release. The lattice area was evaluated by ImageJ and normalized to pre-release area (T0).

2.12. TGF-β1 ELISA

TGF-β1 levels were assayed in the cell supernatants by human TGF-β1 ELISA kit
(Sigma-Aldrich, Saint Louis, MO, USA), as reported in the manufacturer’s instructions.
The adult or aged HDFs were plated at 7000 cells/cm2 and exposed to K at 0.5, 2, or 5% up
to 72 h. Next, the media were centrifuged at 1000× g for 15 min to clarify them from cellular
debris/dead cells. The TGF-β1 concentration was then measured by ELISA kit. The
obtained values were normalized for cell number and expressed as pg/105 cells.

2.13. Statistical Analysis

All data were evaluated by GraphPad Prism version 6.01 (GraphPad Software, San
Diego, CA, USA). To compare the mean values among groups, one-way ANOVA or two-
way ANOVA, followed by Dunnett's post hoc test, was used. Data were expressed as
mean ± SD or mean ± SEM as reported in figure legends. The p values were considered
statistically significant when lowered than 0.05.

3. Results
3.1. Effect of K Formulation on Cell Proliferation of Human Adult and Aged Dermal Fibroblasts
To evaluate the biological effects of K formulation, we used two different cultured
fibroblast models. First, a normal human adult fibroblast cell line, derived from a 47-year-
old female subject, was used at low passage (N = 6–9) to recapitulate the behavior of
normal and adult HDFs and treated with different concentrations of K (0.1, 0.5, 2, 5, and
10%, v/v) for 24, 48, and 72 h. The second used model consisted of HDFs treated with H2O2
25µM for one hour according to the one-step model [23] to obtain aged HDFs and then
exposed to K. As shown in Figure 1A, the two models stood out significantly in the
proliferation rate, evaluated by dynamically monitoring up to 72 h and expressed as cell
confluence. The adult HDFs (controls) showed a gradual growth, while in the aged
fibroblast model, the cell confluence, as expected, did not increase remaining constant
over time. The effect of different concentrations (0.1, 0.5, 2, 5, and 10%, v/v) of K
formulation on adult and aged HDFs was first evaluated with respect to cell proliferation.
As shown in Figure 1A, the proliferation rate of the adult HDFs exposed to increasing
concentrations of K augmented more quickly than that of untreated HDFs, and the rise
was time- and dose-dependent, being statistically significant starting from 0.5%.
Interestingly, when the aged HDFs were exposed to K, the cell confluence enhanced in a
time- and concentration-dependent manner compared to that of the relative untreated
controls; the 5% concentration was the most effective in stimulating the proliferation of
aged HDFs. The analysis of the K formulation effect on the cell number showed a similar
trend in both models (Figure 1B). The cell number of the adult HDFs was always
significantly greater than aged HDFs. The exposure of the adult and aged HDFs to K
formulation caused a marked increase in the cell number compared to that in the relative
controls as incubation time and concentrations increased. Notably, 5% K was the most
effective concentration in inducing an increase in the cell number compared to the
untreated cells in both the adult and aged HDFs (Figure 1B). Representative images from
microscopic observations of adult and aged HDFs treated with 5% K for 72 h confirmed
the increase in the cell number. The aged HDFs appeared shrunken and morphologically
different from the adult HDFs. After the treatment with 5% K, the aged HDF morphology
returned to that more like of the adult HDFs (Figure 1B). This result indicates that K
formulation could counteract the effects of aging on the proliferation rate as well as cell
morphology. Based on the obtained results, the concentrations of K at 0.5, 2, and 5%, being the
most efficacious in stimulating HDF proliferation, were chosen for the following experiments.
Biomedicines 2023, 11, 2410 7 of 16

Figure 1. Effect of K formulation on proliferation rate and cell number of adult and aged HDFs. (A)
Growth curves were analyzed as cell confluence through IncuCyte® instrument and monitored up
to 72 h. Adult and aged HDFs were treated with K formulation at different concentrations (0.1, 0.5,
2, 5, and 10%) in complete medium. Data from one representative out of three independent
experiments are reported as mean ± SD. (B) The cell number of adult and aged HDFs, treated as
described in (A) and stained with trypan blue, was evaluated. Data obtained from three
experiments, in duplicate, are expressed as mean ±SEM. Representative phase-contrast microscopic
images of adult and aged HDFs treated for 72 h with K (5%) are shown (10× magnification). The
comparative analysis of groups of data was performed by the two-way analysis of variance
(ANOVA) followed by Dunnett’s post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

3.2. Effect of K Formulation on β-Galactosidase Activity and Oxidative Stress in Aged HDFs
To investigate the effect of K formulation on the aging of HDFs induced by H2O2, we
analyzed the β-galactosidase (β-gal) activity, known as aging biomarker. The aged cells
showing high β-gal activity are stained blue. As shown in Figure 2A, less than 10% of the
adult control cells were positive for β-gal activity, while more than 90% of the aged control
group revealed the presence of positive β-gal cells. The exposure to K at 0.5, 2, and 5%
was able to decrease the positive cell percentage (82.1% ± 4.6, 55.7% ± 3.1, and 39% ± 2.4,
respectively), counteracting the H2O2-induced aging in a concentration-dependent
manner, and this effect was statistically significant at 2 and 5% (Figure 2A).
The oxidative stress due to the increase in reactive oxygen species (ROS) is strictly
associated with a reduced cell proliferation and subsequent cellular aging [24]. Moreover,
ROS are able to promote collagen degradation, induce the accumulation of elastin, and
inactivate the inhibitors of MMPs, responsible for ECM degradation [25]. The high ROS
level produced by aged HDFs is responsible for the MMP expression increase and the
TGF-β signaling inhibition, promoting dermal aging [26]. To investigate the antioxidant
effect of K formulation in the H2O2-induced cellular aging model, we evaluated the ROS
and malondialdehyde (MDA) levels. As shown in Figure 2B, the intracellular ROS levels
were significantly higher in the aged controls than in the adult controls. The K treatment
significantly reduced them in a concentration-dependent manner constraining H2O2-
induced oxidative stress. Moreover, compared to the adult controls, the secreted MDA
levels were significantly higher in the aged controls, indicating that H2O2-induced injury
caused lipid peroxidation in HDFs. The K formulation treatment significantly reduced the
MDA levels in a time- and concentration-dependent way (Figure 2C). These results
demonstrated that the oxidative stress generated by H2O2 could be strongly counteracted
Biomedicines 2023, 11, 2410 8 of 16

by K treatment. Noteworthy, the 5% concentration was able to restore the ROS and MDA
levels similar to the adult controls.

Figure 2. Effect of K formulation on β-galactosidase activity and oxidative stress in H2O2-induced

aged HDFs. (A) Cellular aging was measured by β-galactosidase staining; the percentage of β-gal
positive cells was determined at different K concentrations. Values are expressed as the means ±
SEM of three independent experiments. Representative images of adult and aged HDFs untreated
(Control) and treated for 72 h with K (5%) are also shown (20× magnification). (B) ROS levels were
evaluated in aged HDFs, using the DCFH-DA assay after treatment. Results are relative to mean
values ± SEM of two experiments performed in duplicate. (C) MDA levels in aged fibroblasts treated
for up to 72 h as described above were assayed in cell supernatants by MDA ELISA kit. Results are
relative to mean values ± SEM of three experiments performed in duplicate. For comparative
analysis of groups of data, one-way or two-way ANOVA followed by Dunnett’s post hoc test was
used (** p < 0.01, *** p < 0.001, **** p < 0.0001).

3.3. Effect of K Formulation on Type I Collagen Synthesis in Adult and Aged HDFs
As type I collagen predominates in the dermis and is also responsible for the tensile
strength of skin tissue, we assessed if the K formulation exposure was able to influence
the collagen I levels. The intracellular collagen I levels, detected by the Western blot and
immunofluorescence (Figure 3A and Figure 3B, respectively), were basically lower in the
aged HDFs than in the adult HDFs, as expected [27]. The Western blot analysis showed
that intracellular type I collagen expression was significantly upregulated in the adult
HDFs exposed to K at all tested concentrations (Figure 3A). Of note, an increase in the
type I collagen levels was observed when the aged HDFs were exposed to K formulation,
with significantly higher levels at 2 and 5%, compared to the aged controls. To confirm
these results, we performed the immunofluorescence analysis (Figure 3B). The adult
HDFs treated with the highest concentration of K displayed more intense and widespread
type I collagen staining than the untreated cells. Of interest, also aged HDFs treated with
5% K showed a substantial increase in staining for collagen I compared to the relative
controls. The levels of collagen secreted by the adult and aged HDFs were assayed in
supernatant, using an ELISA kit (Figure 3C). Preliminary tests excluded the possibility
that ELISA could detect the recombinant polypeptide α1 chain of type 1 collagen in the K
formulation (not shown). In the adult HDFs, K treatment at 0.5, 2, and 5% induced a
significant and dose-dependent increase in extracellular type I collagen, showing an
Biomedicines 2023, 11, 2410 9 of 16

increase percentage of approximately 26–32%, 40–46%, and 48–52%, compared to the

controls, respectively. As expected, the aged HDFs secreted lower basal levels of collagen
than the adult control HDFs, with a reduction of approximately 35–40%. Interestingly,
also in aged HDFs, the exposure to K at 0.5, 2, and 5% induced a significant and dose-
dependent increase in the extracellular collagen levels with an increase percentage of
about ~15–18%, 30–35%, and 30–36%, compared to untreated cells, respectively. After K
treatment, the collagen concentration in aged HDFs were restored and were not
significantly different from those of the controls (Figure 3C). We also evaluated the expression
of P4HA1 protein, a key player in collagen synthesis, in the adult and aged HDFs treated with
increasing concentrations of K formulation for 72 h. As shown in Figure 3D, the basal levels of
P4HA1 in the adult HDFs were higher than those in the aged HDFs. Interestingly, a dose-
dependent increase in P4HA1 expression was observed in the adult HDFs treated with K
when compared to control cells. Following K exposure, an increasing trend of the P4HA1
levels was observed in aged fibroblasts being significant at 2 and 5% (Figure 3D).

Figure 3. Effect of K formulation on type I collagen levels and synthesis in adult and aged HDFs.
(A) Western blot assay for intracellular collagen I was performed on adult and aged HDFs treated
with increasing concentrations of K for 72 h. The values obtained by densitometric analysis were
normalized vs. GAPDH protein. Data are reported as the means ± SEM of two independent
experiments. Images of representative immunoblots are shown. (B) Representative
immunofluorescence images of adult and aged HDFs treated for 72 h with K (5%) and stained with
anti-collagen I antibody (green). Nuclei were counterstained with DAPI (blue). The images are
representative of three independent experiments in duplicate. All images were acquired at 100×
magnification. (C) Collagen levels in fibroblasts treated as described above were evaluated in cell
supernatants, using ELISA kit. Results are relative to mean values ± SEM of three experiments
performed in duplicate. (D) P4HA1 levels were evaluated by Western blot analysis on adult and
aged HDFs treated as described above. In all cases, the comparative analysis of data was performed
using one-way ANOVA followed by Dunnett’s post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001).

3.4. Effect of K Formulation on Fibroblast Contraction and Migration Activity

As already reported [28,29], the fibroblasts basally express the α-SMA protein, which
contributes to cell-generated mechanical tension, and whose expression increases in
activated fibroblasts, improving their contractile activity. To verify whether the increase
in collagen synthesis was associated with fibroblast contraction activity, the expression of
α-SMA in the adult and aged HDFs treated with increasing concentrations of K was
investigated using the Western blot method. As shown in Figure 4A, in the adult HDFs,
the α-SMA protein expression enhanced in a dose-dependent way even if the
upregulation was statistically significant at 5%. Of note, also in the aged HDFs, the
Biomedicines 2023, 11, 2410 10 of 16

treatment induced an increase in the α-SMA levels which was statistically significant at 2
and 5%. With the aim to evaluate the functional effect of K on the contractile machinery
of the adult and aged HDFs, a 3D collagen gel contraction assay was used. The cells were
seeded into a three-dimensional collagen latex, and after latex polymerization, the
complete media containing increasing concentrations of K formulation were added. The
results show that the K treatment at 0.5, 2, and 5% after 24 h led to a marked and
concentration-dependent decrease in the collagen lattice areas with a percentage of
decrease compared to the control group of approximately 5–10%, 44–47%, and 78–82%,
respectively. The obtained results indicate the ability of K to improve the contractility of
HDFs embedded in collagen lattices. Interestingly, the aged HDFs, which showed a lower
contractility function compared to the adult controls of ~20–25%, as evidenced by the
larger disc size, recovered this function with the K treatment. The polycomponent
formulation was able to increase the contractility also in aged HDFs in a concentration-
dependent manner (Figure 4B).

Figure 4. Influence of K formulation treatment on fibroblast activation. (A) Western blot assay for
α-SMA was performed on adult and aged HDFs treated with increasing concentrations of K for 72
h. Following the densitometric analysis, the obtained values were normalized vs. GAPDH protein.
Values are expressed as the means ± SEM of two independent experiments. Representative
immunoblots are also shown. (B) Collagen gel retraction assay was performed on adult and aged
HDF-populated collagen lattices following K treatment. The gel contraction was evaluated by
digital camera, and the lattice area was assessed by ImageJ and normalized to pre-release area (T0).
Normalized area values are reported as mean ± SEM of three independent experiments in duplicate.
Representative images of lattices at T0 and 24 h after treatments are also shown. The yellow dotted
circles mark the lattice’s edges. The comparative analysis of data was performed using one-way
ANOVA followed by Dunnett’s post hoc test (* p < 0.05, *** p < 0.001, **** p < 0.0001).
Biomedicines 2023, 11, 2410 11 of 16

To investigate the effect of K formulation on cell migration in the adult HDFs, the
wound healing assay was performed, and the rate of scratched monolayer closure in the
absence or presence of K at 0.5, 2, or 5% was evaluated by observing the repopulation of
the area between the wound edges at different time points after the lesion. To
quantitatively analyze the effect of K formulation on the closure of the wounded area, the
images obtained by inverted light microscope were acquired at different time points after
scratching and converted to percentage closure (% closure), using a mathematical model
able to automatically calculate the portion of the area occupied by the cells. The analysis
of an in vitro scratch assay showed that the closure percentage at 6, 12, and 24 h increased
in the adult HDFs treated with K compared to the controls in a concentration-dependent
way, and it was significant at 2 and 5%. Since the scratch wound assay was performed in
the presence of the serum, we can conclude that K formulation was able to accelerate the
wound closure, stimulating both cell migration and proliferation of the adult HDFs
(Figure 5). Figure 5 shows the representative microscopic images at 24 h.

Figure 5. Effect of K formulation on cell migration of HDFs. The quantitative results of wound
healing assay in NHDFs treated with increasing concentrations of K. The wound closure was
recorded at 0, 6, 12, and 24 h after the scratch and reported as the wound closure rate (% vs. relative
T0) of scratched monolayers. Showed data are the mean ± SEM of two independent experiments in
duplicate. Two-way ANOVA followed by Dunnett’s post hoc test was used (** p < 0.01, *** p < 0.001,
**** p < 0.0001) to compare the groups of data. Representative images at 24 h of NHDF monolayer
re-epithelialization (the wound edges at T0 are blue dashed lines) are also shown (10×
magnification).

3.5. Effect of K Formulation on Extracellular Secretion of TGF-β1 by Adult and Aged HDFs
Taken into account that TGF-β1 represents a key regulator of fibroblast proliferation,
collagen production, and extracellular matrix renewal in human skin [30,31], we
evaluated the extracellular secretion of TGF-β1 in the adult and aged HDFs treated for up
to 72 h with increasing concentrations of K formulation. After the K exposure, the TGF-β1
levels in the supernatant of the adult HDFs enhanced in a time- and concentration-
dependent way. Of note, in the aged HDFs showing the secreted basal levels of TGF-β1
lower than those in the adult cells, the K treatment induced an increase in the TGF-β1
levels at all tested concentrations (Figure 6). These results suggested that K formulation
could induce the synthesis of the collagen through the TGF-β1 pathway involvement in
the adult and aged HDFs.
Biomedicines 2023, 11, 2410 12 of 16

Figure 6. Effect of K formulation on TGF-β1 levels secreted by adult and aged HDFs. TGF-β1 levels
in adult and aged fibroblasts untreated (Control) or treated for up to 72 h with K at different
concentrations were measured in cell supernatants, using TGF-β1 ELISA kit. Results are from three
experiments performed in duplicate (mean values ± SEM). For comparative analysis of data, two-
way ANOVA followed by Dunnett’s post hoc test was performed (* p < 0.05; **** p < 0.0001).

4. Discussion
In the present study, we investigated the biological effects of an innovative
polycomponent formulation in adult HDFs and in a H2O2-induced aging model of HDFs.
Dermal fibroblasts play a crucial role in the production of ECM components and in
maintaining skin homeostasis. Skin aging is characterized by reduced numbers of
fibroblasts and lower levels of ECM proteins, which decrease elasticity and tonus,
resulting in atrophy, wrinkling, and increased fragility of the skin [5,26]. Skin fibroblasts
are largely used as cellular model for in vitro cellular aging studies. Moreover, fibroblast
H2O2-induced aging is a well-known model of accelerated aging in vitro, as it mimics skin
aging in vivo. Aged fibroblasts are characterized by a cell cycle arrest with long-term loss
of proliferative capacity [32]. Here, we report that K treatment promoted proliferation not
only in the adult cells but also in the aged HDFs, in a time- and concentration-dependent
manner, and improved the changes in the cell morphology counteracting cellular aging.
In addition, the K formulation reduced in the aged HDFs the percentage of cells positive
for β-gal activity, an aging-associated biomarker, in a concentration-dependent manner.
The increased β-galactosidase activity resulted from a rise in the number and size of
lysosomes, which consequently led to an elevated lysosome content in aged cells [33].
Skin aging is closely associated with oxidative stress, a phenomenon characterized
by an imbalance between reactive oxygen species (ROS) and antioxidants. [34,35]. High
levels of ROS oxidize cellular proteins, DNA, and lipids, inducing inflammation,
oxidative damage, and aging in the dermal fibroblasts. The levels of MDA may reflect the
lipid peroxidation level as well as the degree of the consequent cellular damage [36]. In
particular, the MDA is able to destroy the cell membrane integrity, affect the cell structure
and ion transport, and lead to dysfunction of cellular energy metabolism. We then
evaluated the antioxidant activity of the K formulation against H2O2-induced oxidative
stress on aged HDFs. Our findings showed that after treatment with K formulation, the
H2O2-induced upregulation of the intracellular ROS and MDA levels significantly reduced
in a dose-dependent manner, thus effectively exerting antioxidant effects.
One manifestation of human skin aging is losing type I collagen [26]. It is the most
abundant collagen in the skin and is responsible for maintaining tissue architecture. In
addition, collagen I significantly regulates many biological processes, including cell
adhesion, proliferation, and differentiation. Here we show the ability of K formulation to
induce a significant and dose-dependent increase in the intracellular and extracellular
levels of type I collagen in adult or aged HDFs. Collagen synthesis is regulated by several
post-translational modifications that are fundamental for proper stability, assembly, and
Biomedicines 2023, 11, 2410 13 of 16

secretion of triple-helical procollagen, as well as for cleavage of the N- and C-propeptides,

self-assembly of collagen into fibrils, and cross-linking of the fibrils [37]. Prolyl-4-
hydroxylases (P4H), an enzyme that catalyzes the hydroxylation of proline residues of
procollagen, is essential for the folding of newly synthesized procollagen polypeptide
chains into stable triple-helical molecules [38]. Furthermore, the P4H expression levels are
associated with the rate of collagen synthesis; thus, it can be considered a “rate-limiting
enzyme” in collagen production [39]. Considering the crucial role of P4H in collagen
synthesis, we evaluated the ability of the polycomponent formulation to influence its
expression. In the present study, the P4H levels that were basally higher in the adult HDFs
compared to aged HDFs were affected by K formulation, as a significant increase in P4H
was observed in both cell models as K concentrations increased. Considering that P4H is
an essential determinant in initiating collagen biosynthesis, its increased expression level
reflects the correct formation of a functional collagen fibril. We also show evidence that
the K treatment led to a higher expression of α-SMA protein, supporting a link between
the increase in collagen synthesis and activation of fibroblasts. Over the years, it has
become increasingly clear that mechanical forces regulate the synthesis and remodeling
of ECM proteins [40]. The contractility of dermal fibroblasts is closely related to
physiological processes such as wound healing, wrinkling, angiogenesis, and
inflammatory response. Notably, a reduction in collagen expression and ECM
degradation can impair the attachment of dermal fibroblasts within the dermis, resulting
in a change in cellular morphology with less mechanical force [41]. In our experimental
conditions, the contractile activity between the adult and aged HDFs was quite different
under baseline conditions; the treatment with K formulation improved the contractile
activity in both models.
The TGF-β1 pathway plays an essential role in cell contractility, as it initiates the
activation of fibroblasts by inducing the expression of α-SMA [41]. Moreover, TGF-β1 is a
significant regulator of ECM activities, controlling the production of matrix
metalloproteinases (MMPs) and serving as the primary regulator of collagen synthesis.
Several in vitro studies [30,42] have shown that the ECM undergoes progressive
deterioration and fragmentation during skin aging due to reduced collagen transcription
resulting from the attenuated TGF-β1 pathway. Moreover, oxidative damage also
impaired TGF-β1 signaling due to the elevation of ROS that, reducing the type II TGF-β
receptor (TβRII) and SMAD3 protein levels, decrease collagen synthesis [43,44]. Indeed,
aged HDFs showing a significantly lowered TGF-β1 expression express less collagen than
young fibroblasts. Our results show the ability of K formulation to induce a dose- and
time-dependent increase in the TGF-β1 secretion relative to the controls in both models.
Our findings support the notion that this polycomponent formulation could efficiently
activate TGF-β1 signaling in fibroblasts, significantly synthesizing collagen I.
To the best of our knowledge, this is the first study showing in vitro the biological
effects of a polycomponent formulation of non-crosslinked HMW-HA, recombinant
polypeptide of collagen-1 chain, and CMC. Moreover, our results add to the growing
literature indicating that in vitro cellular models may be viable tools for testing the effect
of medical devices without resorting to an animal study. Indeed, according to the
European Directive 63/2010/EU, in vitro methods could significantly contribute to limiting
the use of animal models to evaluate the biological response of medical devices.
An overall interpretation of our results is that the combination of these bioactive
components, being able to improve the biological activities in adult fibroblasts, as well as
counteract H2O2-induced fibroblast aging, could be used to maintain skin homeostasis,
accelerate post-wounding healing, and treat critical age-related skin signs. Additional
research will be needed to deepen further understanding of the involvement of signaling
pathways underlying the polycomponent formulation’s antiaging and antioxidant effects
and evaluate its ability to prevent fibroblast aging.
Biomedicines 2023, 11, 2410 14 of 16

Author Contributions: Conceptualization, F.R.A., F.L. and B.C.; formal analysis, F.L. and P.P.;
investigation, F.R.A., S.A. (Serena Artone), S.A. (Serena Altamura) and A.C.; writing—original draft
preparation, F.R.A., F.L. and B.C.; writing—review and editing, L.D.M., M.G.C., P.P., M.G. and B.C.;
supervision, M.G.C., M.G. and B.C.; project administration, F.L. and P.P.; funding acquisition,
M.G.C., P.P. and B.C. All authors have read and agreed to the published version of the manuscript.
Funding: This research and the APC were funded by Department of Life, Health, and Environmental
Sciences, University of L’Aquila, grants “FFO MeSVA 2021” and “FFO MeSVA 2022”.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets generated and analyzed during the current study are
available from the corresponding authors upon reasonable request.
Acknowledgments: The authors thank Gasperina De Nuntiis (Department of Life, Health, and
Environmental Sciences, University of L’Aquila, L’Aquila, Italy) for excellent technical assistance.
Conflicts of Interest: The authors declare no conflict of interest. The company “Taumedika Srl,
Rome (Italy)” that provided the polycomponent formulation used in the present study had no role
in the experimental design, execution protocols, data interpretation, or manuscript writing.

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