ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 5, pp. 942-957 © Pleiades Publishing, Ltd., 2024.
942
In vitro and in vivo Evaluation of Antifibrotic Properties
of Verteporfin in a Composition of a Collagen Scaffold
Olga S. Rogovaya
1,a
*, Danila S. Abolin
1
, Olga L. Cherkashina
1
, Artem D. Smyslov
1
,
Ekaterina A. Vorotelyak
1
, and Ekaterina P. Kalabusheva
1
1
Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 119334 Moscow, Russia
a
e-mail: rogovaya26f@yandex.ru
Received December 6, 2023
Revised March 5, 2024
Accepted March 31, 2024
Abstract— Extensive skin damage requires specialized therapy that stimulates regeneration processes without
scarring. The possibility of using combination of a collagen gel application as a wound dressing and fibroblast at-
tractant with verteporfin as an antifibrotic agent was examined in vivo and in vitro. In vitro effects of verteporfin
on viability and myofibroblast markers expression were evaluated using fibroblasts isolated from human scar
tissue. In vivo the collagen gel and verteporfin (individually and in combination) were applied into the wound
to investigate scarring during skin regeneration: deviations in skin layer thickness, collagen synthesis, and ex-
tracellular matrix fibers were characterized. The results indicate that verteporfin reduces fibrotic phenotype by
suppressing expression of the contractile protein Sm22α without inducing cell death. However, administration
of verteporfin in combination with the collagen gel disrupts its ability to direct wound healing in a scarless
manner, which may be related to incompatibility of the mechanisms by which collagen and verteporfin control
regeneration.
DOI: 10.1134/S0006297924050146
Keywords: dermal fibroblasts, fibrosis, skin regeneration, verteporfin, YAP/TAZ
Abbreviations: BrdU, 5-bromo-2′-deoxyuridine; BSA,bovine
serum albumin; DPBS, Dulbecco’s phosphate-buffered sa-
line; PFA,paraformaldehyde; VP,verteporfin.
* To whom correspondence should be addressed.
INTRODUCTION
Wound healing is a complex process, which is
commonly considered to be divided into sequential
partly overlapping four phases: hemostasis, inflamma-
tion, proliferation (cell infiltration, angiogenesis, and
re-epithelialization), and maturation/remodelling [1].
At the phase of inflammation activation of immune
cells stimulates secretion of cytokines, which, in turn,
activate migration of fibroblasts, epithelial and endo-
thelial cells to the damaged area. In the wound bed
fibroblasts acquire activated phenotype and are trans-
formed into Sm22 positive myofibroblasts, which are
cells responsible for generation of main components
of extracellular matrix such as fibronectin with extra
domain A, collagen I and III, required for filling-in
defects of connective tissues and scar formation [2-4].
Remodelling phase is the final phase of wound heal-
ing, which could take years. At this stage under nor-
mal conditions architecture of the restored tissue is
close to the structure of normal skin [1].
When the damage is very extensive, specialized
agents are used to stimulate regeneration process.
Tissue equivalents based on the collagen gel are suc-
cessfully introduced into the medicine practice [5].
Biodegradability of collagen and its low immunoge-
nicity make it an optimal base not only for tissue en-
gineering involving design of constructs containing
collagen-based scaffolds and live cells, but also for de-
veloping collagen-based wound dressings [6-8]. Colla-
gen, as a key element of extracellular matrix, affects
all stages of wound healing [9-11]. Collagen hydrogels
partially reproduce properties of extracellular matrix,
have porous structure with a network of protofibrils
enabling migration and colonization of cells, thus fa-
cilitating remodelling of the de novo formed tissue and
wound healing [12]. In some cases, the collagen-based
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BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024
skin substitutes contain biologically active molecules:
growth factors, cytokines or their analogues that stim-
ulate the processes of integration of the tissue equiva-
lent into the damaged organ, regeneration of the adja-
cent tissues, and modulation of immune response.
One of the often complications of skin regenera-
tion, especially in the cases of extensive damages such
as burns requiring specialized therapy, is formation
of hypertrophic scars [13]. In addition to obvious cos-
metic issues, scars disrupt mechanical interactions in
the skin making it more fragile and prone to rapture.
Scars do not contain hair follicles and accompanying
sebaceous glands, which promotes drying of epider-
mis. One of the newest and most promising therapeu-
tic preparations for anti-scar therapy is verteporfin
(VP) [14-17].
Single administration of VP to mice at the early
stages of regeneration initiated the process of scar-
less healing[18]. VP also exhibits bactericidal activity,
which produces beneficial effects on would healing
[16]. Delivery of VP to the damage area for stimulation
of wound healing was successfully performed using
agents based on fibroin [15], polyvinyl [17], and poly-
lactide [14]. We investigated the possibility of using
collagen gel as a VP delivery agent to the wound, in
order to prevent scar formation after complete wound
healing.
The anti-scar properties of VP are considered to
be associated with its ability to inhibit interaction of
the transcription cofactor YAP1 with its targets from
the family of TEAD proteins. Activation of the YAP1
signaling cascades occurs during wound healing both
in epidermis and in dermis [19]. In epidermis it stim-
ulates migration and proliferation of keratinocytes in
the wound bed. Active nuclear YAP1 is associated with
proliferation of fibroblasts and increase of their con-
tractile ability, which is important for wound closing.
Activity of this signaling cascade decreases at the stage
of remodeling. Increase of the duration of activity of
the YAP1 signaling cascade in mice results in forma-
tion of common or hypertrophic scars, at the same
time, its inhibition in fibroblasts at the early stages of
wound healing stimulates regeneration of a fully func-
tional skin with all derivates [20].
Effect of VP on scarring processes were examined
both in vitro and in vivo. For the in vitro studies hu-
man fibroblasts isolated from hypertrophic skin scar
were applied as an analogue of myofibroblasts of the
wound bed. Effect of VP on viability, expression of con-
tractile markers, and contractile ability of fibroblasts
were examined. In the in vivo studies with laboratory
mice, efficiency of VP administration in composition of
collagen gel for stimulation of skin regeneration was
investigated. The following parameters were analyzed:
rate of the wound closure, structure of extracellular ma-
trix, and general morphology of the regenerated skin.
MATERIALS AND METHODS
Isolation and cultivation of dermal fibroblasts.
Cells lines used in the study were isolated from hu-
man skin biopsies obtained in the course of recon-
structing surgeries in the Vishnevsky National Med-
ical Research Center for Higher Technologies with
informed consent of the donors. Experiments with
skin biopsies were performed in accordance with the
protocol approved by the Bioethics Committee of the
Koltzov Institute of Developmental Biology, Russian
Academy of Sciences (IDB) (no. 51 from 09.09.2021).
Biopsy samples were washed with a Hank’s solution
(PanEco, Russia) supplemented with 0.4 mg/ml of
gentamycin (BioFarmGarant, Russia) for disinfection,
next, subcutaneous fat and reticular layer of dermis
were removed mechanically in such a way that the up-
per layer of skin with epidermis was no thicker than
2 mm. Dermis with epidermis were cut into 2-3 mm
bands and incubated in a 2% Dispase solution (Gibco,
USA) for 1 h at 37°C; in the next step epidermis was
removed, dermis was cut by scissors to homogenous
state and incubated in 0.1% collagenase type I solution
(Gibco) for 24 h at 37°C. The obtained mass was cen-
trifuged at 140g for 10 min, precipitate was resuspend-
ed in a DPBS (Dulbecco’s phosphate-buffered saline;
PanEco); this procedure was repeated three times.
To isolate scar fibroblasts normal tissues were re-
moved from the biopsy sample, only regions with pro-
nounced scarring deformation were preserved, which
was followed by incubation of the sample for 1 h in a
2% Dispase solution at 37°C. After removal of epider-
mis, dermis was cut with scissors to homogenous state
and incubated in a 0.1% Liberase solution (Roche,
USA) for 24 h at 37°C. The obtained sample was centri-
fuged at 140g for 10 min; precipitate was resuspended
in DPBS; procedure was repeated 3 times.
Isolated fibroblasts were cultivated in 25 cm
2
cul-
ture flasks an Amniomax-II medium (Gibco) under
conditions of 5% CO
2
and 37°C. Culture medium was
replaced every 2-3 days. After reaching confluency,
cells were detached using a 0.05% trypsin-EDTA solu-
tion (Capricorn, Germany) followed by cultivation in
a DMEM medium (PanEco) supplemented with 10%
fetal bovine serum (Capricorn), 1% Glutamax (Cibco),
1% penicillin-streptomycin (Gibco). Cells after 2-3 pas-
sages were used in the study.
Samples of a healthy skin obtained from three
42-55 years old donors, and samples obtained from
the 33-45 years old donors with scar tissues were used
in the study. Produced cell lines were deposited to the
IDB collection of cell lines for biotechnological and
biomedical studies (general biology and biomedical
section).
Detection of apoptosis. In order to determine
type of the cell death, fibroblasts were detached from
ROGOVAYA et al.944
BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024
Table 1. Sequences of primers used in the study
Gene name Forward primer Reverse primer
GAPDH CCATGTTCGTCATGGGTGTG GGTGCTAAGCAGTTGGTGGTG
HPRT ACCAGGTTATGACCTTGATT AAGTTGGCCTAGTTTATGTT
YAP AGAGAATCAGTCAGAGTGCTCCA TTCAGCCGCAGCCTCTCC
TAZ GGCAATGATTAAACTGGCAACA AGTGAGCCCTTTCTAACCTGG
CTGF TGTGCACCGCCAAAGATG CAGACGAACGTCCATGCTG
CYR61 AAGGAGGCCGTCCTGGTC GGGCTGCATTCCTCTGTGT
EDA-FN CCCTAAAGGACTGGCATTCA CATCCTCAGGGCTCGAGTAG
CD26 AGAAGGAGTATTCAATAAGTGGGAC TACTCTGCTCTGTGGTGGTCT
COL1 AGAAAGGGGTCTCCATGGTG AGGACCTCGGCTTCCAATAG
COL3 CCAGGAGCTAACGGTCTCAG TGATCCAGGGTTTCCATCTC
the surface 1, 3, and 5 days after treatment with VP,
centrifuged at 140g for 5 min, washed once with DPBS,
followed by incubation for 15 min in a solution of
annexin V and propidium iodide using an apoptosis
detection kit PI-AV (BDPharmingen
TM
, USA) according
tomanufacturer instructions. To introduce compensa-
tion coefficient to the results of analysis, samples
stained separately with annexinV and propidium io-
dide were used. Flowcytometry analysis was carried
out with an Attune®NxT flow cytometer (Life Technolo-
gies, USA).
Cultivation of fibroblasts in a collagen gel.
Topre pare gel, a 0.34 M NaOH solution (Khimmed, Rus-
sia) – 6.5%; 7.5% Na
2
CO
3
solution (PanEco) – 3.42%;
10× M199 medium (Gibco)– 9.77%; Glutamax (Gibco)–
0.39%; Hepes (PanEco) – 1.95% collagen type I solu-
tion – 77,97% were mixed on ice. Collagen solution
was prepared from the standard laboratory rat tail
tendons: individual fibers were separated mechani-
cally and dissolved in a 0.1% acetic acid, final concen-
tration– 2 µg/ml. Gel aliquots (400 µl) containing cell
suspension (150 × 10
3
cells perml) were inoculated into
wells of 24-well plate (Corning, USA) and incubated
at 37°C for polymerization. Culture medium (500 µl)
was added on top of the gel. Gel was separated from
the culture well walls, which facilitated unlimited con-
traction of the gel by cells.
One day after the gel formation, VP (Sigma-Al-
drich) solution in a culture medium at concentration
0.1 µg/ml was added to experimental wells followed by
incubation with the cells for 24 h followed by washing
the gel once with a Hank’s solution and immersing the
gel into a regular culture medium.
Quantitative PCR analysis. RNA was isolated
with the help of a RNAzol reagent (Sigma-Aldrich, Ger-
many) according to the manufacturer’s instructions.
AQuantiTect Reverse Transcription Kit (Qiagen, USA)
was used for genomic DNA removal and perform-
ing reverse transcription. One µg of RNA was used
for reverse transcription reaction. Real-time quan-
titative PCR was carried out using a 5× qPCRmix-HS
SYBR+LowROX reagent kit (Evrogen, Russia) and a
LightCycler96 amplifier (Roche). Reaction protocol:
10 min at 95°C, 1 cycle; next 45 cycles including 20 s
at 95°C, 20 s at 60°C, 30 s at 72°C. All experiments
were performed in three biological and technical rep-
licates. Amount of product in each sample was cal-
culated using the 2
–ΔΔCq
method and normalized to
expression of GAPDH and HPRT. Data on the graphs
are presented as mean ± standard deviation. Sequenc-
es of the primers used in the study are presented
in Table1.
Incorporation of BrdU label and determination
of the cell cycle stage. Fibroblasts (500 × 10
3
cells)
were seeded into 25 cm
2
culture flasks (Corning) and
cultured for 1 day. Next, VP was added at concentra-
tion 0.1 µg/ml. Concentration of VP was determined
previously (unpublished data) with consideration of
the literature data [18] and cultivation was continued
for 1 day followed by removal of VP with replacing
culture medium. Next, a BrdU label (5-bromo-2′-deoxy-
uridine; Sigma-Aldrich) was introduced on the day 1,
3, and 5of cultivation to final concentration 30 µM fol-
lowed by 2-h incubation. Cells were detached, washed
with DPBS, and fixed in a 70% ethanol solution for 1 h
at 4°C. Next cell was precipitated by centrifugation for
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BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024
Table 2. Antibodies used in the study
Antibodies Manufacturer, cat. no. Dilution Fluorophore
Primary anti-BrdU rat IgG Abcam, ab6326 1/100 –
Primary goat IgG against Sm22α Abcam, ab10135 1/100 –
Primary rabbit IgG against YAP1 Abcam, ab52771 1/100 –
Primary rabbit IgG against YAP1 Cell Signaling Technology, D8H1X 1/100 –
Primary rabbit IgG against collagen I+III Imtek (Russia), RAP c13 1/20 –
Primary goat IgG against P-cadherin R&D Systems, AF761 1/20 –
Primary rat IgG against E-cadherin Abcam, ab11512 1/20 –
Secondary donkey IgG against rabbit IgG Invitrogen, 32790 1/1000 Alexa 488
Secondary donkey IgG against goat IgG Life Technologies, A11056 1/1000 Alexa 546
Secondary goat IgG goat against rat IgG Life Technologies, A11081 1/1000 Alexa 546
2 min at 1000g; 0.5 ml of 2 M HCl (Khimreaktiv, Rus-
sia) containing 0.5% Triton X-100 (MP Biomedicals,
USA) was added to the precipitate and incubated for
30 min at room temperature. Cell was precipitated, su-
pernatant was removed, and cells were resuspended
in 0.5 ml of 0.1 M sodium tetraborate (Khimmed, Rus-
sia). The mixture was incubated for 2 min and, next,
washed once with 150 µl of DPBS/1% BSA (bovine se-
rum albumin; PAA, Austria), and next incubated with
50 µl of a primary antibody solution in DPBS with ad-
dition 0.5% Tween 20 (MP Biomedicals) and 1% BSA
for 1 h at room temperature. Cells were washed once
with DPBS and incubated in a solution of secondary
antibodies for 30 min at room temperature (manu-
facturers and dilutions of antibodies are presented
in Table 2). Next, cells were resuspended in 0.5 ml
of DPBS containing 10 µg/ml of RNAse A (Fermentas,
Canada) and 20 µg/ml of propidium iodide solution
(Sigma-Aldrich) followed by 30-min incubation in the
dark. Cells stained with relevant secondary antibodies
and non-stained cells were used as controls. Flow cy-
tometry was carried out using an Attune® NxT flow
cytometer.
Immunofluorescent staining of collagen gels.
Gels were fixed with a 4% solution of paraformalde-
hyde (PFA) (Sigma-Aldrich) for 40 min, washed once
with DPBS, and permeabilized in a DPBS solution con-
taining 1% Tween 20, 1% Triton X-100, and 5% BSA
for 1 h at room temperature. Next, primary antibod-
ies at concentrations shown in Table 2 were added
and incubated for 12 h at 4°C. Next, the samples were
washed with permeabilizing solution for 2 h followed
by addition of the corresponding secondary antibod-
ies conjugated with fluorophores (Table 2) and 12-h
incubation at 4°C. Nuclei were stained with DAPI (Bi-
otium, USA) at concentration 1 µg/ml. Imaging and
image analysis was carried out with the help of an
Olympus IX 73 fluorescence microscope (Olympus,
Japan).
Surgical procedures. Male C57Bl6 mice (n = 24)
at the age of 8 weeks were used in the study. All exper-
iments were conducted with animal under general an-
esthesia in accordance with the International Guiding
Principles for Biomedical Research Involving Animals
[21, 22]. Animal were kept with free access to water
and feed.
Mice were divided into the following groups: Con-
trol(n = 6), VP(n = 6), Gel(n = 6), and Gel + VP(n = 6).
Animals were anesthetized with isoflurane VetFarm,
Russia). Hair was shaved between the shoulder blades,
and surgical area was disinfected with 70% alcohol.
Ketoprofen solution (50 mg/ml, Ellara, Russia) was in-
jected into a surgical area. A 5-mm diameter circle was
cut from all layers of the skin. To prevent rapid con-
traction, a silicone ring (splint) with diameter corre-
sponding to the wound size was applied, the ring was
glued with a BF-6 glue (Tulskaya Farmatsevticheska-
ya Fabrika, Russia) and fixed in place with 8 surgical
stiches using a nonabsorbable suture (Resolon, Germa-
ny). The wound was rinsed and 250 µl of collagen gel
(‘Gel’ group), 100µl of VP solution in PBS with concen-
tration 1 mg/ml (‘VP’ group), or 250 µl of collagen gel
with VP (‘Gel + VP’ group at the rate 100 µg of VP per
mouse) were administered into the wound of animals
in experimental groups. In the ‘Control’ group 100 µl
of PBS solution with added DMSO (dilution 1/1) was
ROGOVAYA et al.946
BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024
applied to the wound. Wounds were covered with a
Tagaderm film (3M, Germany) and fixed with a ban-
dage (Omniplast, Germany).
Collagen gel for introduction into wounds was
prepared the same way as in the case of fibroblast
cultivation. Gel (250 µl) was placed into the wells of a
48-well plate (Corning) and incubated at 37°C for po-
lymerization.
State of wounds in animals was examined every
other day, and additional sutures were applied to the
silicon rings if required. Wounds were photographed
once a week.
All abovementioned agents were introduced once
more into the wounds after 3 days. On the day 21 of the
experiment the animals were euthanized, post-mortem
7-8 mm diameter biopsy samples including entire skin
thickness were taken from the wounds.
Handling of wound biopsy samples and histo-
logical staining. Wound biopsies were embedded into
a Tissue-Tek medium (Sakura, Japan) forming cryob-
locks by incubation first in nitrogen vapour and, next,
immersing them into liquid nitrogen several times
for 3-5 s until completely frozen; samples were stored
at–70°C. Cryosections of the samples with 8 µm thick-
ness were prepared with a Leica CM1950 cryostat
(Leica,Germany).
Sections were fixed in a 4% PFA solution, stained
with a hematoxylin-eosin stain (BioVitrum, Russia) ac-
cording to the manufacturer’s protocol, and embedded
into a Bio Mount medium (BioOptica, Italy). Imaging
of the stained samples was carried out with a Keyence
BZ-9000 microscope (Keyence,Japan).
For immunofluorescence analysis, sections were
fixed in a 4% PFA solution, incubated in a solution of
primary antibodies containing 5% BSA, 1% Triton X-100,
and 1% Tween 20 for 18 h, primary antibodies were
next removed by washing in DPBS, and incubated with
solution of secondary antibodies for1.5 h. Imaging of
the samples was carried out with a Leica Thunder mi-
croscope (Leica). List of the antibodies and their con-
centration are shown in Table 2.
Analysis of collagen fibers architecture. Archi-
tecture of collagen fibers was analyzed in the samples
stained with hematoxylin-eosin. Dermis in the wound
area was imaged with 40× magnification, the obtained
images were analyzed with the help of CurveAlign and
CT-FIREalgorithms [23,24].
The CurveAlign algorithm analyzes the degree of
alignment of collagen fibers in an image and con-
structs heat map of the fiber orientation; CT-FIRE uses
curvelet transformation to suppress noise in the im-
age and determine boundaries of collagen fibers, and,
next, extracts information and analyzes parameters of
individual collagen fibers (length, width, straightness,
angle) [23, 24]. The obtained data were statistically
processed.
Statistical analysis. Statistical data processing
was carried out with the help GraphPadPrism 9 pro-
gram. Prior to selection of statistical criterium each
data set was analyzed for normality of distribution
(Shapiro–Wilk test, Kolmogorov–Smirnov test, D’Agos-
tino–Pearson combined test, Anderson–Darling test).
In the case of normal data distribution differences be-
tween groups were analyzed with the help of one-way
analysis of variance (ANOVA) with post hoc Tukey test
for multiple comparisons, or with the help of two-way
ANOVA supplemented with the Sidak test for pairwise
comparisons. In the case of deviation from normal dis-
tribution differences between the data were analyzed
with the Kruskal–Wallis test and post hoc Dunn’s test
for multiple comparisons. Presence of outliners in the
data was determined with the help of Dixon’s Q test.
The data obtained with the help of algorithms for
analysis of collagen fiber architecture were processes
using the OriginPro 2022 software. Principal compo-
nent analysis was performed based on the results ob-
tained with the help of these algorithms. The graphs
show 95% confidence intervals for each group of data.
K-means clustering was performed to identify similar
data sets.
RESULTS
Effect of VP on viability of normal and scar-de-
rived fibroblasts. At the first stage of the study, we
evaluated effect of VP on viability of normal and
scar-derived human skin fibroblasts. For this pur-
pose, the cell cultures were treated with VP solution
for 24 h and, next, number of live and dying cells was
determined with the help of propidium iodide and an-
nexin V staining after 1, 3, and 5 days of cultivation
(Fig. 1, a and b). Live cells were identified based on
lack of staining with propidium iodide and annexinV.
The level of proliferation was determined based on the
number of cells with incorporated BrdU (Fig. 1c).
No statistically significant changes in the number
of live/dying cells after treatment with VP were ob-
served. However, treatment with VP significantly de-
creased proliferation in the normal and scar-derived
fibroblasts one day after treatment. Three days af-
ter treatment the levels of proliferation between the
control (norm and scar) and experimental groups
(norm + VP, scar + VP) groups differed not very sig-
nificantly, but the decreasing trend was observed.
On the fifth day after treatment the scar-derived fi-
broblasts exhibited tendency for increased level of
proliferation in the ‘VP’ group in comparison with
the control group. Interestingly enough, prolifer-
ation decreased in the groups not treated with VP
from day 1 to 5. This is associated with the contact
inhibition in the course of reaching the confluence.
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BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024
Fig. 1. Evaluation of the number of dead and proliferating fibroblasts of different types (norm and scar) under the action of VP.
a)Fraction of live cells after treatment with VP. b)Fraction of dying cells after treatment with VP. c)Number of proliferating
cells. Data are presented as a mean ± standard deviation. Statistically significant differences: **p≤0.01 and ****p≤0.0001.
ROGOVAYA et al.948
BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024
Fig. 2. Analysis of cell cycle of normal(norm) and scar-derived(scar) fibroblasts after VP treatment. Data are presented as
amean ± standard deviation.
Considering that the analysis did not reveal any dif-
ferences in the cell death, but showed differences in
proliferation, we decided to analyze of the cell cycle
(Fig.2).
No statistically significant changes in distribution
of cells according to the cell cycle phases after treat-
ment with VP were observed. Nevertheless, this dia-
gram allows suggesting that the decrease in prolifera-
tion is due to delay in the G1 phase.
Effect of VP on behaviour of dermal fibroblasts
in a collagen gel. Fibroblasts placed into the collagen
gel after just one day form contacts with the colla-
gen fibrils acquiring elongated phenotype typical for
this type of cells. At the same time fibroblasts initiate
contraction of the gel. Incubation in the presence of
0.1 µg/ml ofVP was carried out for 24 h in the half of
the prepared gels; culture medium with added DMSO
was used as a control. Evaluation of the gel size was
estimated 3, 5, and7days after treatment(Fig. 3a). Re-
sults of the analysis demonstrated that VP initially de-
creases the degree of gel contraction in both types of
investigated fibroblasts. The scar-derived fibroblasts
preserved their fibrotic properties during cultivation
and contracted gel to a greater degree in comparison
with the normal fibroblasts. The analyzed gels visual-
ly contained the same number of cells; hence, intensi-
ty of contraction was determined by the characteris-
tics of fibroblasts.
Expression of Sm22α, one of the markers of my-
ofibroblasts mediating their contractile capacity, was
investigated using immunofluorescent techniques [25-
27]. Expression of the elements of the YAP1 signaling
pathway was also evaluated, because VP is an inhibi-
tor of this cascade [28,29](Fig.3).