Connective Tissue Research

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/icts20

Minoxidil decreases collagen I deposition and

tissue-like contraction in clubfoot-derived cells:
a way to improve conservative treatment of
relapsed clubfoot?

Jarmila Knitlova, Martina Doubkova, Martin Plencner, David Vondrasek,

Adam Eckhardt, Martin Ostadal, Jana Musilkova, Lucie Bacakova & Tomas
Novotny

To cite this article: Jarmila Knitlova, Martina Doubkova, Martin Plencner, David Vondrasek,
Adam Eckhardt, Martin Ostadal, Jana Musilkova, Lucie Bacakova & Tomas Novotny (2021)
Minoxidil decreases collagen I deposition and tissue-like contraction in clubfoot-derived cells:
a way to improve conservative treatment of relapsed clubfoot?, Connective Tissue Research,
62:5, 554-569, DOI: 10.1080/03008207.2020.1816992

To link to this article: https://doi.org/10.1080/03008207.2020.1816992

© 2020 The Author(s). Published by Informa Published online: 20 Sep 2020.

UK Limited, trading as Taylor & Francis
Group.

Submit your article to this journal Article views: 5945

View related articles View Crossmark data

Citing articles: 2 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=icts20
CONNECTIVE TISSUE RESEARCH
2021, VOL. 62, NO. 5, 554–569
https://doi.org/10.1080/03008207.2020.1816992

Minoxidil decreases collagen I deposition and tissue-like contraction in clubfoot-

derived cells: a way to improve conservative treatment of relapsed clubfoot?
Jarmila Knitlova a*, Martina Doubkova a,b*, Martin Plencner a, David Vondrasek a,c
, Adam Eckhardt a
,
Martin Ostadal d, Jana Musilkova a, Lucie Bacakova a, and Tomas Novotny b,e
a
Department of Biomaterials and Tissue Engineering, Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic;
b
Second Faculty of Medicine, Charles University, Prague, Czech Republic; cFaculty of Physical Education and Sport, Charles University, Prague,
Czech Republic; dDepartment of Orthopaedics, First Faculty of Medicine, Charles University and Na Bulovce Hospital, Prague, Czech Republic;
e
Department of Orthopaedics, Masaryk Hospital, Usti Nad Labem, Czech Republic

ABSTRACT ARTICLE HISTORY

Aim: Clubfoot is a congenital deformity affecting the musculoskeletal system, resulting in con­ Received 27 February 2020
tracted and stiff tissue in the medial part of the foot. Minoxidil (MXD) has an inhibitory effect on Accepted 25 August 2020
lysyl hydroxylase, which influences the quality of extracellular matrix crosslinking, and could
KEYWORDS
therefore be used to reduce the stiffness and to improve the flexibility of the tissue. We assessed
Relapsed clubfoot;
the in vitro antifibrotic effects of minoxidil on clubfoot-derived cells. congenital idiopathic talipes
Methods: Cell viability and proliferation were quantified by xCELLigence, MTS, and LIVE/DEAD equinovarus; CTEV; fibrosis;
assays. The amount of collagen I deposited into the extracellular matrix was quantified using minoxidil; collagen type I
immunofluorescence with subsequent image segmentation analysis, hydroxyproline assay, and
Second Harmonic Generation imaging. Extracellular matrix contraction was studied in a 3D model
of cell-populated collagen gel lattices.
Results: MXD concentrations of 0.25, 0.5, and 0.75 mM inhibited the cell proliferation in a
concentration-dependent manner without causing a cytotoxic effect. Exposure to ≥0.5 mM MXD
resulted in a decrease in collagen type I accumulation after 8 and 21 days in culture. Changes in
collagen fiber assembly were observed by immunofluorescence microscopy and nonlinear optical
microscopy (second harmonic generation). MXD also inhibited the contraction of cell-populated
collagen lattices (0.5 mM by 22%; 0.75 mM by 28%).
Conclusions: Minoxidil exerts an in vitro inhibitory effect on the cell proliferation, collagen
accumulation, and extracellular matrix contraction processes that are associated with clubfoot
fibrosis. This study provides important preliminary results demonstrating the potential relevance
of MXD for adjuvant pharmacological therapy in standard treatment of relapsed clubfoot.

Introduction relapses5. In the case of relapsed clubfoot, there is also the

Idiopathic talipes equinovarus (clubfoot, CTEV) is one of
the most common congenital orthopedic deformities
affecting the musculoskeletal system of the lower limb.
Two methods are frequently chosen for use in the treat­
ment of clubfoot. The Ponseti method is used worldwide
and involves serial manipulations combined with a specific
cast application technique, which can be supplemented by
percutaneous Achilles tenotomy, followed by foot abduc­
tion bracing to prevent relapses1. The less widely used
French method consists of daily physiotherapy, stimulation
of the muscles around the foot, and temporary foot immo­
bilization with elastic and non-elastic adhesive taping2.
Idiopathic clubfeet are mainly treated by the Ponseti
method with excellent results3,4; however, the long-term
successful outcome of the treatment can be spoiled by
possibility of surgical therapy, which can be applied as
described in the literature6. However, studies of the long-
term results of these surgeries have reported several com­
plications, such as stiffness of the ankle, arthritis, muscle
weakness, pain, and residual deformity3,7.
Although several hypotheses about clubfoot pathogen­
esis have been proposed, there is still no clear understand­
ing of its cause and its early progression on a genetic,
molecular, and cellular level. Current hypotheses cover, e.
g., environmental factors, including in utero fetus
positioning8 or enteroviral infection9; developmental fac­
tors like abnormal muscle insertions10 or vascular
deficiencies11; and also genetic factors such as homeobox
gene abnormalities12. This range of hypotheses suggests
that clubfoot pathology is probably of multifactorial origin.

CONTACT Martina Doubkova Martina.Doubkova@fgu.cas.cz Institute of Physiology of the Czech Academy of Sciences, Prague 14220, Czech
Republic
*J. Knitlova and M. Doubkova contributed equally to this work as first authors.
© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-
nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built
upon in any way.

Older histological and electron microscopy studies
have suggested the existence of fibrotic changes in
tissue biopsies extracted from the clubfoot deformity­
13–15
. More recently published proteomic and molecu­
lar studies have revealed changes in the amount of
collagen type I, III, VI, and profibrotic cytokines, such
as transforming growth factor (TGF-β) and platelet-
derived growth factor (PDGF) in tissue from clubfoot
medial side16–18 and also in m. gastrocnemius19. These
studies have therefore confirmed the presence of fibro­
sis in clubfoot. The fibrotic tissue foci of the clubfoot
medial side are located between medial malleolus, sus­
tentaculum tali, and the navicular bone. This area is
sometimes called “disc-like” tissue20, due to its extreme
stiffness and its rigid nature, which is caused by hyper­
production of extracellular matrix (ECM) components.
Accumulation of ECM proteins, with collagens being
the most abundant component, is characteristic of
fibrosis. Collagen production by highly proliferating
fibroblasts, tissue architecture, and contraction ability
is regulated by growth factors, especially by TGF-β21.
Therefore, collagen formation and growth factors reg­
ulating fibroblast behavior would be major targets in
the development of an antifibrotic therapy.
Research on possible antifibrotic therapies for various
fibroproliferative diseases is relatively widespread (for a
review, see22). For some therapies, licensed pharmacolo­
gical agents are already available. In Dupuytren’s disease,
for example, local administration of clostridial collagenase
into the collagen-rich tissue contractures in fingers con­
tributes to the digestion of excess collagen and to tissue
release23. Although some possible targets have been sug­
gested in idiopathic clubfoot, very little attention has been
given to the idea of using pharmacological agents in the
clubfoot tissue, with the exception of studies by Li et al.17
and Poon et al.24. The application of antifibrotic sub­
stances directly into the fibrotic tissue on the clubfoot
medial side could reduce tissue contracture, and thus
could accelerate and/or improve the outcomes of conser­
vative treatment in relapsed patients.
Minoxidil is an FDA-approved drug for treatment of
hypertension and hair loss25–27. Its main antifibrotic
potential lies in the decrease in lysyl hydroxylase
mRNA expression, which effectively reduces the activ­
ity of the enzyme28,29. Lysyl hydroxylases act on specific
lysine residues in the procollagen alpha chains, which
later in the collagen biosynthesis process determine the
type and the quantity of crosslinks in newly-formed
collagen fibers. For the proper biosynthesis and struc­
tural stability of collagen fibers, it is essential to estab­
lish collagen crosslinks; however, the quality of mature
collagen fibers can vary significantly. Specifically, lysyl
hydroxylase 2 isoform activity (for a review, see30) has
EFFECT OF MINOXIDIL ON CLUBFOOT-DERIVED CELLS 555
been found to be responsible for the increased forma­
tion of the crosslinks described in various fibrotic tis­
sues, including hypertrophic scars31,32 and Dupuytren’s
disease33. In contrast to clostridial collagenase, which is
used as a therapeutic agent in Dupuytren’s disease,
minoxidil does not degrade collagen fibers already
formed in the ECM. This is an important factor for
the safety of its medicinal application. Its effects do not
entirely prevent abnormal collagen accumulation; how­
ever, they may help to lessen it by forming ECM that is
more susceptible to degradation. In this way, minoxidil
could have a positive impact on the stability and the
stiffness of the whole ECM.
The aim of our study was to assess the in vitro
antifibrotic effects of minoxidil as a candidate for the
development of potential adjuvant treatment for
relapsed clubfoot. Such antifibrotic treatment may
reduce the stiffness and the contraction of the tissue,
and thus may improve the outcomes of relapsed club­
foot treatment.
Methods
Acquisition of biological samples
The primary cell cultures used in this study were iso­
lated from tissue samples from 11 patients (8 boys and
3 girls in total; median age of 5 years, SD = 2, Table 1)
with idiopathic congenital talipes equinovarus (CTEV,
clubfoot). All of these patients had been treated in
accordance with the Ponseti method1, as described in
Table 1. Tissue samples were acquired during surgeries
indicated in the case of a clubfoot relapse; the area of
interest was the fibrotic “disc-like” tissue localized
between medial malleolus, sustentaculum tali, and the
navicular bone on the medial side of the foot.
The acquisition of samples and the cell experiments
were approved by the ethical committee. Parents and/
or legal guardians of all sample donors gave their
informed consent. All procedures were performed in
compliance with the Declaration of Helsinki (1934).
Isolation, cultivation, and characterization of cells
Tissue samples were temporarily stored in cell culture
media, and were processed within one hour after surgical
excision. The samples (average weight 500 mg) were then
cut into very small pieces and were enzymatically digested
for at least 3 hours in an enzyme solution (1 ml per 50 mg of
tissue) at 37 °C, 5% CO2 on a shaker installed within an
incubator. The enzyme solution consisted of clostridial
collagenase type 3 (298 U/ml) and neutral protease
(0.560 U/ml) (both Worthington Biochemical
556 J. KNITLOVA ET AL.

Table 1. Detailed data of 11 patients (sample donors) with idiopathic congenital clubfoot.

Dimeglio Age during Casting Clubfoot Foot

clubfoot sample (number of Compliance with family (left/
Patient Gender classification acquisition plasters) Type of surgery Previous surgeries abduction bar anamnesis right)
1 M III 5 6 Mc Kay Achilles tenotomy no 0 L
2 F III 3 5 Mc Kay Achilles tenotomy no 0 R
3 M IV 1* 10 Mc Kay None no 0 L
4 M II 5 3 Fowler Achilles tenotomy yes 0 R
5 F IV 4 6 Mc Kay Achilles tenotomy no 0 R
6 M III 5 5 Mc Kay None no 1 (mother) R
7 M III 5 6 Mc Kay Achilles tenotomy no 0 R
8 M IV 1* 10 Mc Kay None no 0 R
9 M III 10 5 Mc Kay Achilles tenotomy no 0 R
10 F IV 1* 10 Mc Kay Achilles tenotomy no 0 R
+ retenotomy
11 M III 7 5 McKay Achilles tenotomy yes 0 L
Total 8 1x II; Median = 5 Median = 6 Mc Kay = 90%; 8 in 11 patients 9x no; 1x 3x L;
males; 6x III; Fowler = 10% 2x yes 8x R
3 4x IV
females
* Patients whose parents were noncompliant, were otherwise unable to maintain proper treatment by application of an abduction bar or failed to attend
scheduled examinations.

Corporation, USA), and 2.5% antibiotic–antimycotic solu­
tion (Sigma-Aldrich, USA) dissolved in Dulbecco’s mod­
ified Eagle’s Medium (DMEM; Sigma-Aldrich, USA). The
cell suspension was then seeded into poly-L-lysine-coated
tissue culture flasks (75 cm2; TPP, Techno Plastic Products,
Switzerland) and was cultivated in a 37 °C incubator with
5% CO2 saturation in 20 ml of the DMEM medium, which
was supplemented with 20% of fetal bovine serum (FBS;
Gibco, Life Technologies, USA) and gentamicin (40 μg/mL,
LEK, Slovenia). After approximately 10 days, the cells
reached confluence and were passaged.
A basic characterization of the primary cell culture
was performed in order to identify a subpopulation of
cells associated with fibrosis (myofibroblasts,
fibrocytes)34. The cell cultures were characterized by a
NovoCyte Flow Cytometer (ACEA Biosciences, USA)
for the presence of CD90, CD45, and CD34 markers,
collagen type I (Col1), smooth muscle α-actin (αSMA),
and myosin heavy chain (SMM MYH11). To discrimi­
nate the cell types more precisely, the cells were also
immunofluorescently stained for Col1, αSMA, SMM,
desmin, vimentin, and von Willebrand factor35–37.
In order to evaluate the time required for cell spread­
ing and the cell population doubling time, the cells in the
1st passage were fixed in cold (−20 °C) 70% ethanol for
10 min at room temperature (RT), were washed with
Phosphate-Buffered Saline (PBS; Sigma-Aldrich Co.,
USA), and were fluorescently stained for 1 hour (RT,
in the dark) with Hoechst #33,258 (5 µg/ml in PBS;
94,403, Sigma-Aldrich, USA) to visualize the cell nuclei,
and with Texas Red C2 Maleimide (20 ng/ml in PBS;
Sigma-Aldrich Co., USA; red stain) to visualize the cell
membrane and the cytoplasmic proteins. The fluores­
cence signal was evaluated by an Olympus IX51
epifluorescence microscope with a DP74 camera (both
Olympus Corp., Japan). The microphotographs that
were obtained (10 per well) were analyzed in ImageJ
FIJI software (v.1.52 n; open-source)38. The cell spread­
ing area was measured during the first 24 hours of
culture (after 1, 3, 5, and 24 hours). The doubling time
was determined in the exponential phase of cell growth
(i.e. on day 1 and 3 after seeding) according to the
following equation:
t t0
DT ¼ log2
logNt logNt0
Time intervals after seeding are represented by t0
(earlier interval) and by t (later interval), while Nt0
and Nt represent the number of cells on the sample at
these intervals.
Preparation of the minoxidil solution
Minoxidil (MXD) powder (M4145, Sigma-Aldrich,
USA) was dissolved in 96% ethanol (EtOH) to a
119 mM stock solution, was aliquoted, and was stored
frozen at −20 °C. Working concentrations were diluted
immediately prior to each experiment in DMEM sup­
plemented with 10% FBS.
Cell cultivation in the presence of minoxidil
The cells for the experiments were seeded into 24-well
plates (TPP, Techno Plastic Products, Switzerland) in
1 ml of DMEM with 20% FBS per well. For the Live/
Dead viability/cytotoxicity assay, the cells were seeded
at an initial density of 2 × 104 cells per well (ca. 10,730
cells/cm2). For the MTS assay, immunofluorescence
staining of collagen, the hydroxyproline assay, qPCR,

and nonlinear optical microscopy (SHG imaging), the
cells were seeded at an initial density of 3 × 104 cells per
well (ca. 16,094 cells/cm2). The cell number and viabi­
lity before seeding were determined using a Vi-CELL
XR Analyzer (Beckman Coulter, USA). After 24 hours,
the cells were supplied with fresh DMEM with 10% FBS
and either MXD (final concentrations of 0 mM,
0.25 mM, 0.5 mM and 0.75 mM, where 0 mM is
regarded as untreated cells and is marked as Control),
or corresponding volume concentrations of 96% etha­
nol (vehicle equivalent control). Addition of MXD was
considered as the start day (day 0) of treatment for all
experiments. In experiments focused on the production
and deposition of collagen type I, all samples were also
supplemented with L-ascorbic acid-2-phosphate (Asc-
2P; final concentration of 50 µg/ml). The medium was
replaced by the same amount of fresh DMEM with 10%
FBS with MXD or vehicle control and Asc-2P on each
5th day of culture.
The cell seeding numbers and the cultivation condi­
tions for experiments in the xCELLigence system and
in the collagen gel lattices differ from all other experi­
ments and are described in detail in the appropriate
method sections.
Cytotoxicity testing
For the cytotoxicity tests, the cells from the first passage
were grown to 80% confluence (ca. 5 day) before seed­
ing for a given test.
xCELLigence—real-time cellular analysis (RTCA)
The xCELLigence system (ACEA Biosciences, USA)
was used to determine the nontoxic concentration of
minoxidil. This system allows real-time monitoring of
cell attachment, spreading, and changes in cell num­
bers, reflecting the cell viability in response to the
addition of MXD, by measuring the electrical impe­
dance. This impedance is generated by cells contacting
the gold microelectrode arrays incorporated on the
bottom of 96-well plates. The changes in the cell-micro­
electrode contact, i.e. the changes in cell coverage of the
well bottom, are reflected by the relative change in the
impedance in time, which is calculated by RTCA soft­
ware (ACEA Biosciences, USA) as the Cell Index (CI).
For monitoring CI, the cells were seeded into a 96-well
plate (E-Plate 96; ACEA Biosciences, USA) at a density
of 3.5 × 103 cells per well (ca. 10,870 cells/cm2), and in
200 µl of DMEM medium supplemented with 20% FBS.
The cells were left to adhere for 30 minutes before the
plate was mounted into the xCELLigence system and
before monitoring began. After 24 hours of monitoring,
the cells were treated for 48 hours either with 0, 0.25,
EFFECT OF MINOXIDIL ON CLUBFOOT-DERIVED CELLS 557
0.5, 1, and 2 mM MXD or with corresponding concen­
trations of 96% ethanol (EtOH vehicle controls) in
200 µl of DMEM with 10% FBS. After this period, the
cells were supplied with fresh DMEM with 20% FBS
and were monitored to assess cell recovery from MXD
treatment. Since monitoring the CI (which reflects only
the coverage of the well bottom with cells) cannot
distinguish between the size of the cell spreading area
and the number of cells, optical monitoring was also
included in the analysis. A microscopic evaluation of
the cell morphology was performed in two time inter­
vals, i.e. immediately after the removal of MXD
(72 hours of culture) and after an additional 92-hour
recovery period (164 hours of culture).
MTS assay
The cell metabolic activity in the presence of MXD was
assessed using an MTS colorimetric assay (G3582,
CellTiter 96 AQueous One Solution Cell Proliferation
Assay, MTS; Promega Corporation, USA). This assay
offers quantification of the activity of mitochondrial
enzymes, and it therefore also provides indirect infor­
mation on cell proliferation and viability.
The cells for the experiments were seeded into 24-
well plates at an initial density of 3 × 104 cells per well
(16,094 cells/cm2), and were cultivated and treated as
described above (i.e., in the section “Cell cultivation in
the presence of minoxidil”). The MTS assay was per­
formed according to the manufacturer’s protocol on
day 1 and on day 4 of the treatment. The reduction in
MTS was measured using a Synergy HT Microplate
Reader spectrophotometer (BioTek Instruments, USA)
at 490 nm (with a reference wavelength at 650 nm). The
metabolic activity of the MXD-treated samples and the
samples treated with EtOH vehicle control was com­
pared with the metabolic activity of untreated control
cells.
Live/dead assay
In order to further test the effect of MXD, the cell
viability and proliferation was also evaluated by fluor­
escence staining on day 1, day 4, and day 8 after the
start of MXD treatment, using the LIVE/DEAD
Viability/Cytotoxicity kit for mammalian cells (L3224,
ThermoFisher Scientific, USA). This method distin­
guishes between living and dead cells by simultaneous
staining in green, for living cells, and in red, for dead
cells. The cells were seeded into 24-well plates at an
initial density of 2 × 104 cells per well (ca. 10,730 cells/
cm2), and were cultivated and treated as described
above (i.e., in the section “Cell cultivation in the pre­
sence of minoxidil”) for 8 days. The fluorescence signal
was visualized by an Olympus IX51 epifluorescence
558 J. KNITLOVA ET AL.
microscope with a DP74 camera (both Olympus Corp.,
Japan). The microphotographs that were obtained (10
per well) were analyzed in ImageJ FIJI software
(v.1.52 n; open-source)38 by counting the living
(green) and dead (red) cells. Viability was then deter­
mined as the percentage of the living cells in the cul­
ture. The cell numbers were expressed as cell
population densities per cm2.
Collagen production and deposition
Immunofluorescence of collagen type I—short-term
collagen deposition
Cells for immunofluorescence staining were seeded as
described above (i.e., in the section “Cell Cultivation in
the presence of minoxidil”) on microscopic glass cover­
slips inserted into a 24-well plate and cultivated for
8 days. After that, the cells were fixed in cold (−20 °
C) 70% ethanol for 10 min at room temperature (RT),
were washed with PBS, and were then treated with 1%
bovine serum albumin and 0.1% Triton X-100 in PBS
for 30 min at RT in order to block the nonspecific
binding sites for antibodies. Subsequently, a primary
mouse monoclonal anti-collagen type I antibody
(1:200 in PBS; C2456, Sigma-Aldrich, USA), which
recognizes the native conformation of collagen I, was
applied overnight at 4 °C. After washing with PBS, a
secondary antibody Alexa Fluor 488-conjugated F(ab’)2
fragment of goat anti-mouse IgG (1:400 in PBS; A1107,
Invitrogen, USA) was added for 1 hour at RT together
with Hoechst #33,258 (5 µg/ml in PBS; 94,403, Sigma-
Aldrich, USA) to stain the cell nuclei. The fluorescence
signal was visualized by an Olympus IX51 epifluores­
cence microscope with a DP74 camera (both Olympus
Corp., Japan). The microphotographs that were
obtained (10 per well) were analyzed by ImageJ FIJI
software (v.1.52 n; open-source)38, using a pixel-based
segmentation tool. The amount of deposited collagen
type I was expressed as a percentage of the stained
collagen area in the whole area of a single image, and
was normalized to the number of cells on that image.
The data are presented as a percentage of fluorescently
stained collagen area per cell.
Hydroxyproline assay—long-term collagen
deposition
Cells were seeded as described above and were culti­
vated for 21 days. First, the cells were lysed in RIPA
buffer (R0278, Sigma-Aldrich, USA), and a Bradford
assay was performed to measure the total protein con­
tent. Then the amount of hydroxyproline was deter­
mined by the Hydroxyproline Colorimetric Assay Kit
(K555, BioVision, USA), according to the
manufacturer’s instructions, and it was measured
using a VersaMax Absorbance Microplate Reader
(Molecular Devices, USA) at 560 nm. The amount of
hydroxyproline in a sample is widely used as a direct
measure of native collagen. The data are presented as
the relative concentration of hydroxyproline to the total
protein content per sample.
Second harmonic generation imaging
A second harmonic generation (SHG) signal was
obtained in a confocal microscope in order to deter­
mine the presence of properly structured collagen type
I fibers. The cells were seeded on microscopic glass
coverslips inserted in a 24-well plate and cultivated as
described above. On day 21 of cultivation, the cover­
slips with cells were taken out of the plate, and the cells
were visualized using a 63x water immersion objective
(HC PL APO CS 2 63x/1.20 Water), mounted on a
Leica DMi8 inverted microscope with a Leica TCS
SP8 X confocal unit (Leica Microsystems, Germany).
A Chameleon Discovery TPC pulsed femtosecond laser
(Coherent Inc., USA; 860 nm tunable output, 80 Mhz,
1.6 W) was used to generate SHG and autofluorescence
signals. Leica non-descanned HyD detectors and a 430/
24 band-pass filter were used for collecting SHG sig­
nals, and Leica non-descanned HyD detectors and a
610/75 band-pass filter were used for collecting auto­
fluorescence signals.
Real-time PCR
Cells were seeded as described above. Collagen type I
expression was measured using real-time PCR (qPCR).
On day 3 and day 7 of MXD treatment, the cell RNA
was isolated using the Total RNA Purification Micro
Kit Plus (48,500, Norgen Biotek, Canada) according to
the manufacturer’s instructions. RNA in a concentra­
tion of 300ng/µl was used for reverse transcription into
cDNA by the ProtoScript First Strand cDNA Synthesis
Kit (E6300, New England BioLabs, USA), using the
oligo-dT primers that were supplied. The reaction ran
in a T-Personal Thermocycler (Biometra, Germany).
The real-time PCR was performed using 5xHOT
FIREPol Probe qPCR Mix Plus (ROX) (08–14-00008,
Solis BioDyne, Estonia) and TaqMan Gene Expression
Assays (Life Technologies, USA) labeled with the FAM
reporter dye specific to target human gene collagen
type I alpha chain (COL1A1; Hs00164004_m1, ampli­
con length 66 bp), and a reference gene glyceraldehyde-
3-phosphate dehydrogenase (GAPDH; Hs02758991_g1,
amplicon length 93 bp). A final reaction was conducted
in the Viia 7 Real-time PCR System (Applied
Biosystems, USA) in a total reaction volume of 20 μl
and the following cycle parameters: incubation at 95 °C

(10 min), followed by 40 cycles of 95 °C (15 sec) and
60 °C (1 min). The relative mRNA expression was
quantified by the 2−ΔΔCt method. The data were nor­
malized according to the gene expression in the control
sample (Ctrl, calibrator).
Cell-mediated contraction of collagen gel lattices
The CytoSelect 24-Well Cell Contraction Assay Kit-
floating matrix model (CBA-5020, Cell Biolabs, USA)
was used to study the effect of MXD on the cytoskele­
ton of fibroblast-like cells, and on the contraction of
their ECM in vitro. Collagen gel lattices were prepared
according to the manufacturer’s instructions in a 24-
well anti-adhesive plate with 385 × 103 cells per 0.5 ml
of collagen gel per well, and were incubated in DMEM
with 10% FBS and either MXD (0-1 mM) or corre­
sponding concentrations of 96% ethanol (EtOH vehicle
control) with daily changes of 50% of the media for a
freshly mixed solution. To exclude the possibility of
changes in the pure collagen gel lattices (without addi­
tion of cells) due to MXD treatment, all MXD concen­
trations were also tested with unpopulated lattices.
Unpopulated lattices with no treatment served as nega­
tive controls where no change should occur. After
2 days of cultivation, the changes in the sizes of the
gel lattice areas were photographed and were measured
using ImageJ FIJI software (v.1.52 n; open-source)38.
An MTS test was carried out at the end of the experi­
ment in order to verify the viability of the cells inside
the collagen lattices.
Statistical analysis
A statistical analysis was performed in SigmaStat 4.0
(Systat Software Inc., USA). Data from all experiments
were analyzed either by ANOVA with Dunnett’s post
hoc test or by Kruskal-Wallis ANOVA on Ranks with
Dunnett’s post hoc test (specified in the respective
figure caption). P-values < 0.05 were considered statis­
tically significant. All graphs were created using
GraphPad Prism 6 (GraphPad Software, USA).
Table 2. Characterization of the cells derived from the relapsed clubfoot, with emphasis on the presence of cell types associated
with fibrosis.
Cell type CD34 CD45
Fibroblasts - -
Myofibroblasts - -
Fibrocytes + +
Vascular smooth muscle cells NT NT
* ± sign = positivity/negativity of markers for the cell to be identified as a particular type. NT = marker not tested. dim+ = marker is at least dim positive.
EFFECT OF MINOXIDIL ON CLUBFOOT-DERIVED CELLS 559
Results
Clubfoot primary cell culture characterization
First we performed a basic characterization of the pri­
mary cell culture with the aim to identify cell subpopula­
tions that are associated with fibrosis (myofibroblasts,
fibrocytes)34. The culture contained mainly fibroblasts
(96%) with an admixture of myofibroblasts and fibro­
cytes (<3%), vascular smooth muscle cells (<0.1%)
(Table 2), while no endothelial cells (identified by von
Willebrand factor positive staining) were present in the
culture. Rarely, adipocytes were identified on the basis of
their morphology. However, a pure fibroblast culture
was obtained in the fourth passage. Therefore only the
first, the second, or the third passage was used for the
experiments. We refer to these cells as fibroblast-like.
The time required for cell spreading and the cell
population doubling time were evaluated in the first
passage. The cell spreading area was increasing gradu­
ally from 1 hour to 24 hours after seeding, when it
reached its maximum (ca 1940 μm2). The estimated
doubling time of 30.4 ± 1.08 hours (mean ± SEM)
was determined in the exponential phase of cell growth
(from days 1 to 3 after seeding).
Is minoxidil a safe agent for fibroblast-like cells
derived from fibrotic relapsed clubfoot tissue?
We started by testing the maximum final concentration of
MXD that can be added to clubfoot fibroblast-like cells
without causing a direct cytotoxic effect. The progression
of cell spreading and proliferation throughout the experi­
ment was estimated by the xCELLigence system. Electrical
monitoring and also optical monitoring revealed that with
an increasing concentration of MXD, the cell spreading
and proliferation were suppressed dramatically (Figure 1).
The control cells were also grown in DMEM with 10% FBS
during the period of MXD treatment. This serum supple­
mentation in the media proved to be enough to support the
growth and the viability of the cells, and at the same time,
no protective effect of serum against MXD was apparent.
Some cells became rounded in the presence of 1 mM MXD,
and even more cells were rounded in 2 mM MXD, as
Analyzed markers
SMM
CD90 Col1 αSMA MYH 11 vimentin desmin % in culture
+ + - - + - ~ 96%
+ + + - + - < 3%
+ + - - + - < 3%
NT dim+ + + - + < 0.1%
560 J. KNITLOVA ET AL.

Figure 1. (a). Real-time analysis of the effect of minoxidil (MXD) on the growth of fibroblast-like cells using the xCELLigence system.
(b) Morphology of native cells. The cells were seeded 24 hours before the start of the minoxidil (MXD) treatment and were observed
immediately after the MXD treatment period (corresponding to a 72-hour culture time interval; 1b first row) and at the end of the
recovery period, which followed after the removal of MXD (corresponding to a 164-hour culture time interval; 1b second row). The
effect of EtOH vehicle controls was in general negligible. EtOH max indicates the effect of EtOH vehicle control, corresponding to the
maximum MXD concentration (2 mM), on the cells. Pictures of native cells, Olympus IX51 microscope, DP71 digital camera, obj. 20x,
bar = 50 µm.

observed under a phase contrast microscope (Figure 1(b),
first row). However, this effect was reversible, as all cells
recovered during subsequent cultivation in a fresh DMEM
medium with 20% FBS (without MXD), and reached con­
fluence eventually (Figure 1(b), second row). Nevertheless,
we excluded the 2 mM MXD concentration from further
experiments. Instead, we added a concentration of
0.75 mM MXD to better discriminate the gradual effects
of MXD on the cells.
In order to determine the possible cytotoxicity of the
MXD concentrations that were applied, we performed a
standard MTS metabolic assay (Figure 2), and we inter­
preted the results according to the International
Standard for testing in vitro cytotoxicity (International

Figure 2. MTS assay. Dose-response relationship for the effect of minoxidil (MXD) concentration on cell proliferation and viability,
expressed in terms of cell metabolic activity after 1 and 4 days of treatment. Data presented as a percentage value of the metabolic
activity (A490) of cells treated with MXD or cells treated with the ethanol (EtOH) vehicle control, in comparison to the untreated
control (regarded as the 100% value). Mean ± SEM. Day 1: One way ANOVA, Dunnett’s test; statistical significance compared to the
control: *** p < 0.001. However, the decrease in metabolic activity on day 1 did not reach the limit stated by the ISO standard, and it
is therefore considered not to be biologically significant. Day 4: Kruskal-Wallis ANOVA on Ranks, Dunnett’s test; statistical significance
compared to the untreated control: ** p < 0.01 (specifically: MXD 0.75 mM, p = 0.002; MXD 1 mM, p ≤ 0.009).
 EFFECT OF MINOXIDIL ON CLUBFOOT-DERIVED CELLS 561

Figure 3. LIVE/DEAD assay. The effect of minoxidil (MXD) concentration on (a) the cell proliferation rate and (b) cell viability after 1,
4, and 8 days of MXD treatment. The cells were seeded 24 hours before the start of the treatment at an initial density of ca. 10,730
cells/cm2. Proliferation data presented as mean ± SEM. Day 1 and Day 4: One way ANOVA, Dunnett’s test; without statistical
significance. Day 8: One Way ANOVA, Dunnett’s test; statistical significance compared to the untreated control (Control): * p < 0.05,
** p < 0.01 (specifically: MXD 0.50 mM, p = 0.01; MXD 0.75 mM, p = 0.003). Viability data presented as the median of the percentage
of living cells to the total cell number; the whiskers depict maximum and minimum values. One way ANOVA; without statistical
significance.

Organization for Standardization, 2009)39, as an assess­
ment of cell viability (measured by the cell metabolic
activity) after 24 hours of cultivation with the tested
substance. According to the ISO standard, an agent
that reduces cell viability to less than 70% of an
untreated control sample is considered to have cytotoxic
potential. In the case of MTS, this is indicated by an
appropriate change in the measured absorbance at
490 nm (A490).
The MTS assay was applied to the cells not only on
day 1, but also on day 4 of MXD treatment, in order to
acquire more complex information about the possible
cytotoxic effects. On day 1, none of the MXD concen­
trations reduced the cell metabolic activity to less than
70% of the control, although both 0.75 mM MXD and
1 mM MXD showed a statistically significant decrease in
A490 compared to the control. In the case of 0.75 mM
MXD, the decrease was to 83.5% of the control value
(p < 0.001), and in the case of 1 mM MXD, the decrease
was to 85.7% of the control value (p < 0.001) (Figure 2).
However, these decreases did not reach the limit stated
by the ISO standard, and they can therefore be consid­
ered biologically non-significant. Similarly, on day 4,
statistically significant decreases in A490 compared to
the control were detected only for MXD 0.75 mM and
1 mM. The cell viability decreased to 53.9% of the
control value in the case of 0.75 mM MXD
(p = 0.002), and decreased even to 36% of the control
value in the case of 1 mM MXD (p ≤ 0.009). The
metabolic activity of cells treated with 0.5 mM MXD
dropped to 71.5% of the control value, but the statistical
test used for the data analysis did not classify this
decrease as statistically significant (Figure 2). In the
samples treated with ethanol (EtOH) vehicle equivalents
to the tested MXD concentrations, the cell metabolic
activity never dropped below 82%, and this drop showed
no statistical significance. Although the ISO standard
does not specifically state an acceptable reduction in
MTS-measured viability for 4 days of cultivation, the
reduction after treatment with 1 mM MXD to 36% of
the control was so enormous that we excluded this
concentration from further experiments.
The MTS assay, i.e. a test of the activity of mitochondrial
enzymes, is stated to indirectly assess cell viability and, at
the same time, to reflect cell proliferation. However, this
assay does not distinguish whether the cells are dying or are
only proliferating more slowly. We therefore performed a
third type of cytotoxic test, the LIVE/DEAD assay, for
concentrations of 0–0.75 mM MXD. In all samples, includ­
ing those treated with the tested MXD concentrations and
the corresponding EtOH vehicle controls, the number of
viable cells per sample never dropped below 98%. However,
the growth in cell numbers per cm2 between the experi­
mental intervals declined slowly over time (Figure 3). It
even appeared that cells treated with 0.50 mM MXD and
with 0.75 mM MXD almost reached the stationary phase of
cell growth during the time period between day 4 and day 8.
These results clearly indicate that minoxidil has an anti­
proliferative effect, rather than a cytotoxic effect, on fibro­
blast-like cells. The influence of the EtOH vehicle
equivalent on cell proliferation and viability in these experi­
ments was negligible in comparison with the untreated
Controls.
The cytotoxicity and proliferation tests revealed that
MXD reduces cell proliferation in a dose-dependent
manner, while it does not have a direct cytotoxic effect.
On the basis of these results, we chose three concentra­
tions for further testing of their antifibrotic activity
(0.25, 0.5, and 0.75 mM MXD). These concentrations
are safe for the cells and do not inhibit cell proliferation
completely. In the following experiments, the antipro­
liferative effect of minoxidil was taken into account
and, when applicable, the results that were obtained
were normalized to the number of cells on the sample
562 J. KNITLOVA ET AL.

or in the area of interest, or to the total protein content
in each sample (depending on the type of test).
Does minoxidil reduce the amount of collagen type
I in the extracellular matrix of relapsed clubfoot-
derived cells?
We investigated the assumed antifibrotic effect of min­
oxidil by measuring collagen type I deposition into the
ECM after 8 days of cultivation. An analysis of micro­
photographs from fluorescence microscopy showed
that a concentration of 0.25 mM MXD had no signifi­
cant effect on collagen deposition (median = 43.9x10−3,
IQR = 0.024, p = 0.135). However, both of the higher
concentrations significantly reduced the collagen con­
tent: 0.5 mM MXD (median = 14 x10−3, IQR = 0.009,
p < 0.001) and 0.75 mM MXD (median = 9 x10−3,
IQR = 0.009, p < 0.001) in comparison with the control
(median = 79x10−3, IQR = 0.038) (Figure 4).
The collagen I production pathway is naturally
regulated at various levels on its way from mRNA
expression via procollagen secretion to the formation
of mature collagen fibers, all in reaction to the current
cell state and condition. We investigated whether min­
oxidil treatment can influence procollagen type I
expression in reaction to the reduced cell population
density or in reaction to the reduced amount of col­
lagen type I in the ECM. We measured the level of
procollagen type 1 alpha-1 chain (a procollagen type I
structural component, encoded by COL1A1 gene)
mRNA by real-time PCR (Figure 5). We found that
there was no concentration-dependent effect of

Figure 4. The short-term collagen deposition is lower in the minoxidil (MXD)-treated samples after 8 days of treatment, as
represented by the smaller area covered by fluorescently stained collagen. (a) Changes in collagen type I deposition visualized by
immunofluorescence staining. The amount of collagen type I (green signal, upper panel) after treatment with increasing concentra­
tions of MXD decreased gradually, but not in proportion to the lower number of cells in culture (blue signal, lower panel, merged
with the upper panel). Olympus IX51 microscope, obj. 10x, bar = 200 µm. (b) Percentage of the fluorescently stained collagen area
expressed per single cell. Image analysis of microphotographs obtained via fluorescence microscopy. Data presented as a box plot
with a median line, the outer edges represent the 1st and the 3rd quartile, the whiskers depict maximum and minimum values.
Kruskal-Wallis ANOVA on Ranks, Dunnett’s test; statistical significance compared to the untreated Control: *** p < 0.001.

Figure 5. COL1A1 gene expression after 3 and 7 days of minoxidil (MXD) treatment. The data are normalized according to the gene
expression in the untreated control sample (Control, calibrator; represented by the dashed line), and are presented as a box plot
with a median line, the outer edges represent the 1st and the 3rd quartile, the whiskers depict maximum and minimum values.
Kruskal-Wallis ANOVA on Ranks, Dunnett’s test; without statistical significance.
minoxidil on collagen type 1 alpha 1 chain mRNA
expression after 3 or 7 days of cultivation in the pre­
sence of MXD. This result indicates that the inhibitory
effect on collagen is realized downstream from the
COL1A1 expression. The influence of the vehicle
equivalent (EtOH) on the cells in these experiments
was not statistically significant (data not shown).
To assess the long-term effect of minoxidil treatment on
collagen type I deposition, we measured the content of
collagen type I in ECM after a 3-week cultivation period
by means of a hydroxyproline assay. As in the case of short-
term cultivation, only 0.5 mM and 0.75 mM MXD had a
significant effect on collagen deposition into the ECM:
0.5 mM MXD (median = 1.04x10−3, IQR = 0.46x10−3,
p ≤ 0.004), 0.75 mM MXD (median = 0.848x10−3,
IQR = 0.234x10−3, p ≤ 0.004) in comparison with the
untreated control (median = 2.50x10−3, IQR = 0.89x10−3)
(Figure 6(a)).
After a 3-week cultivation period, the collagen
fibers were so abundant that it was not possible to
visualize them by conventional fluorescence micro­
scopy. We therefore used second harmonic generation
(SHG) imaging, i.e. label-free confocal microscopy, to
acquire qualitative information on the collagen type I
molecular assembly. The representative pictures in
Figure 6(b) depict the decrease in the SHG signal
generated by properly assembled collagen fibers with
increasing concentration of minoxidil, while they also
illustrate the reduction in cell numbers after long-term
cultivation.
The LIVE/DEAD assay performed in parallel
revealed that there is no cytotoxicity, even within
3 weeks of cultivation and repeated treatment with
MXD in all concentrations (data not shown here).
EFFECT OF MINOXIDIL ON CLUBFOOT-DERIVED CELLS 563
Can minoxidil reduce the tissue-like contraction of
the extracellular matrix caused by relapsed
clubfoot-derived cells?
In order to explore the effect of minoxidil on cell-
mediated contraction of ECM, we used 3D collagen
gel lattices as an in vitro model of ECM. The area of
the gel seeded with cells without the addition of MXD
(control) was the most contracted, and was therefore
regarded as a 100% value of the area shrinkage. The
percentage values of the MXD-treated samples express
by how many percent the gels are less contracted in
comparison to the control (Figure 7(a)). No significant
changes were observed in cell-populated gel lattice
areas, when only the vehicle equivalent (EtOH) was
added (data not shown here). Gels without cells, with
or without MXD treatment, did not exhibit any shrink­
age at all during the experiments.
A concentration of 0.25 mM MXD (SEM = 0.02,
p = 0.067) had no significant effect on the contraction
of cell-populated collagen gel lattices, as the shrinkage of
the gel area was only 9% less than the shrinkage of the
untreated control. However, the gel lattices incubated in
0.5 mM, 0.75 mM, and 1 mM MXD were significantly
less contracted. The contraction was by 22%, by 28% and
by 35% less than in the control (SEM = 0.27, 0.28, and
0.32, respectively, p ≤ 0.001) (Figure 7(a)).
The 3D model of the collagen gel lattices resem­
bles the environment of connective tissue ECM
more closely than the situation when cells are
grown in a monolayer. We repeated the MTS tests
for cells in a gel with a concentration of 0.25–1 mM
MXD because of the different culture conditions in
these experiments. MTS confirmed that the cells
564 J. KNITLOVA ET AL.

Figure 6. (a) The long-term collagen type I deposition after 3 weeks is lower in the minoxidil(MXD)-treated samples than in the
untreated control, as represented by a lower relative hydroxyproline content in these samples. Data presented as a box plot with a
median line, the outer edges represent the 1st and the 3rd quartile, the whiskers depict the maximum and minimum values. Kruskal-
Wallis ANOVA on Ranks, Dunnett’s test; statistical significance compared to the untreated control: ** p < 0.01, *** p < 0.001
(specifically: MXD 0.5 mM, p < 0.001; MXD 0.75 mM, p = 0.004). (b) SHG imaging of collagen type I. The decrease in the SHG signal in
clubfoot fibroblast-like cells treated with minoxidil demonstrates its effect on the correct structural assembly of collagen type I
(upper panel, green signal). The persistent effect of minoxidil concentration-dependent suppression of cell proliferation is still
apparent after 3 weeks of cultivation by the lower number of cells in the samples (autofluorescence of cells, lower panel, red signal).
Leica DMi8 confocal microscope, obj. 63x water immersion, bar = 50 µm.

thrive in these conditions, as the viability of all Discussion

samples did not drop below 95% of the control
(Figure 7(b)).
The influence of minoxidil on the cell-mediated con­
traction of collagen gel lattices populated by fibroblast-
like cells is illustrated in representative pictures taken
after 2 days of treatment (Figure 7(c)).
Minoxidil (MXD) has previously been tested in vitro in
human dermal fibroblasts and in keratinocytes,40–42
and also in vivo in mice with induced pulmonary
fibrosis43. However, its antifibrotic effect on clubfoot
cells, derived directly from the contracted part of the
tissue, has been assessed for the first time in this study.
 EFFECT OF MINOXIDIL ON CLUBFOOT-DERIVED CELLS 565

Figure 7. (a) The contraction of collagen gel lattices by clubfoot fibroblast-like cells after 2 days of minoxidil (MXD) treatment is
presented as a percentage of the decrease in the gel lattice contraction in comparison with the control sample. Mean ± SEM. One
Way ANOVA, Dunnett’s test; statistical significance in comparison with the untreated control: *** p < 0.001. (b) Effect of minoxidil
(MXD) concentration on the proliferation and viability of cells in collagen lattices, expressed in terms of the cell metabolic activity
after 2 days of treatment (MTS assay). Data presented as a percentage value of the metabolic activity (A490) of cells treated with
MXD to the untreated control cells (regarded as 100%). Mean ± SEM. One Way ANOVA, Dunnett’s test; without statistical
significance. (c) Pictures representing the changes in collagen gel areas after 2 days of MXD treatment.

Admittedly, there are some limitations to our investi­
gation that should be addressed here. As all our experi­
ments were performed in vitro, it is uncertain how well
our findings would be translated into in vivo conditions.
The extracellular matrix formed in vitro is produced
by cells which may lack important factors, e.g. those
coming from blood vessels or secreted by other cell
types in the surrounding tissue. An exact simulation
of all these signals in vitro, however, is almost impos­
sible to achieve. Although fibrotic markers could be
modeled by adding profibrotic cytokines (e.g. TGF-β,
PDGF) to the cell culture, it still would not fully repre­
sent the complex interplay among soluble factors and
cells in vivo. It is also likely that cells originating from
relapsed clubfoot tissue lose their fibrotic characteristics
with prolonged in vitro cultivation. Taking this into
consideration, we performed all experiments only
within the first, the second, or the third passage of
cells isolated from each tissue sample. For example,
we found that the amount of myofibroblasts in culture
declined, and this cell type became almost absent in the
third passage in vitro.
We are also aware that the selected effective concen­
trations of MXD relate to the in vitro situation. For
potential adjuvant treatment of relapsed clubfoot, local
delivery directly into the area of medial side contracted
tissue seems to be the most logical administration
route. However, in the literature there are no data on
MXD pharmacokinetics in connective tissue for admin­
istration of this type. In vitro testing is the first step in
analyzing the antifibrotic actions of MXD in clubfoot,
and we consider that animal models of fibrosis could be
introduced in the not-so-distant future. Two knockout
mouse models of clubfoot were recently established.44,45
Further research is necessary in order to achieve the
desired sustainable antifibrotic effect in vivo, probably
with MXD bound to a biomacromolecular carrier with
affinity to the affected tissue. Such a targeted carrier can
increase the efficacy of the treatment, can simulta­
neously reduce possible side-effects, and can lessen
the immunogenicity of the drug (for a review, see46).
Another limitation of our study is the relatively
small number of patients used as tissue sample donors
(n = 11; Table 1). Operative treatment of idiopathic
relapsed clubfoot is very rare, so there are limited
opportunities to obtain tissue samples from this exact
region of the foot. Nevertheless, samples obtained from
operative treatment have provided a good opportunity
to study the stiff tissue of clubfoot.
We have shown in this study that MXD is not
cytotoxic to clubfoot-derived fibroblast-like cells, but
that it lowers their proliferation in a dose-dependent
manner. This finding is in agreement with the previous
work of Pinnell and Murad29,40. The administration of
1 mM MXD in Pinnell and Murad’s study caused
almost complete cessation of skin fibroblast growth in
subconfluent cultures, but no cytotoxic effect was
reported. Tissue fibrosis is associated with hyperproli­
feration of fibroblasts, and a moderate decline in cell
proliferation is therefore even desirable.
566 J. KNITLOVA ET AL.
The main consequence of fibrotic processes in ECM is a
massive net accumulation of collagen. Antifibrotic strate­
gies involve inhibiting either growth factors or cytokines,
driving the cells into contractile phenotype, which is
accompanied by massive collagen production (for a review,
see47), or targeting the biosynthetic pathway of collagen
itself (for a review, see48). To the best of our knowledge,
only one study to date has examined the use of antifibrotics
in clubfoot-derived cells in vitro17. Li et al.17 also reported
decreased cell proliferation in addition to decreased col­
lagen type III mRNA expression in clubfoot-derived cells
when they were treated with neutralizing antibodies to
TGF-β and PDGF. However, Li et al.17 do not provide
data on collagen deposition into the matrix. We took a
different strategy, and targeted the post-translational mod­
ifications/processing which occurs during collagen
biosynthesis.
We observed that concentrations of 0.5 mM and
0.75 mM MXD lowered the deposition and the structural
maturation of collagen type I into ECM in both studied
time intervals (after 8 days and after 3 weeks of MXD
treatment). This took place without the procollagen type I
alpha-1 chain mRNA (encoded by COL1A1 gene) expres­
sion being affected. The expression of procollagen type I
alpha-2 chain mRNA (encoded by COL1A2 gene), i.e. the
other chain needed for forming a collagen triple-helix,
was also not affected by MXD in human dermal fibro­
blasts and cells from keloid scars in vitro42. However, this
may not be the case for all types of cells and tissues. In
contradiction to our findings for clubfoot, the expression
of COL1A1 was reduced by MXD in vivo in the lung
tissue of mice with bleomycin-induced fibrosis43. The
exact effect of MXD on the mRNA expression of genes
encoding procollagen chains should therefore be further
tested on a larger number of relapsed clubfoot samples.
Minoxidil reduces the levels of individual lysyl hydro­
xylase isoforms to a varying extent (LH1≫LH2>LH3).42
To reduce fibrosis, it would be advantageous to inhibit
predominantly the hydroxylation of telopeptidic lysin resi­
dues, which is catalyzed by lysyl hydroxylase isoform 2b
(LH2b). However, no specific inhibitor of LH2 is currently
available, despite efforts that are being made to identify
such compounds.49
Another important posttranslational modification of
collagen, which takes place after hydroxylation, is the
glycosylation of hydroxylysyl residues. It is possible that
by limiting the supply of hydroxylysin residues, minox­
idil indirectly inhibits glycosylation of collagen and
thus modulates collagen fibrillogenesis at different in
its biosynthesis. It has been shown that the LH3 iso­
form of lysyl hydroxylase also possesses a modest glu­
cosyltransferase and galactosyltransferase activity.50
However the LH3 isoform is affected only to a limited
extent by minoxidil.42
Interestingly, the ability of MXD to reduce collagen
deposition was also observed in bleomycin-induced
lung fibrosis in mice, and was suggested to be at least
partially linked to the downregulation of TGF-β/Smad3
signaling.43 As Merl-Pham et al.51 have recently shown
in their study on human lung fibrosis, TGF-β can also
modulate collagen glycosylation. It is probable that the
decrease in collagen deposition in MXD-treated sam­
ples observed in the present study is due to the effect of
MXD on some steps in the posttranslational modifica­
tion of collagen.
The observed changes in the diameters of cell-popu­
lated collagen gel lattices indicated that minoxidil can
also modulate cell-matrix mechanical forces, and thus it
can lessen the contraction in a 3D tissue-like model of
ECM. We speculate that the inhibition of collagen gel
contraction is not due to reduced collagen deposition
or decreasing cell numbers. Shrinkage of cell-populated
collagen gels was observable within hours after it had
been prepared, but inhibition of these processes by
MXD does not occur so quickly. Dallon and Ehrlich52
reviewed the possible mechanism of collagen lattice
contraction, and pointed out that after seeding within
the gel, the cells attach and elongate. This process
generates forces that translocate the collagen fibrils
and cause shrinkage of the collagen gel. A microscopic
examination of the cells in specific intervals (72 and
164 hours of culture) during real-time xCELLigence
monitoring showed that MXD affects the cell morphol­
ogy in a dose-dependent manner (Figure 1(b)). With
increasing MXD concentration, the cells gradually
changed their morphology from elongated well-spread
cells with long processes to less-elongated smaller cells,
and often to completely rounded cells at 2 mM MXD
(Figure 1(b)). It is probable that the effect of MXD on
collagen gel contraction is associated with changes in
cell morphology. Another possible explanation comes
from the work of Shao et al.43, who proved that minox­
idil inhibits the signal transduction of TGF-β/Smad3
and reduces collagen formation, thus reducing the lung
fibrosis previously induced by bleomycin in mice. The
association between TGF-β and increased cell contrac­
tility is well known, and its regulation by minoxidil in
clubfoot-derived cells would be of great importance. It
has also been reported by Priestley et al.41 that minox­
idil decreased the secretion of glysosaminoglycans
(GAG) in skin fibroblasts, an important component of
ECM in direct interaction with collagen. This subse­
quently resulted in decreased contractility of collagen
gel lattices.

Conclusion
Minoxidil inhibits the cell proliferation of clubfoot
fibroblast-like cells in vitro in a concentration-depen­
dent manner, but does not cause cytotoxicity per se.
Concentrations smaller than or equal to 1 mM can be
considered safe for use with clubfoot fibroblast-like
cells in vitro. However, a concentration of 0.5 mM
seems to be enough to elicit significant changes in
extracellular matrix collagen content. Minoxidil lowers
both short-term and long-term deposition and the
structural maturation/assembly of collagen type I fibers.
At the same time, it has no considerable influence on
the expression of the COL1A1 gene, indicating that the
inhibitory effect on collagen type I production and on
structural maturation is realized downstream from
COL1A1 expression. In addition, minoxidil reduces
the cell-mediated contraction of cell-populated collagen
gel lattices in a concentration-dependent manner.
Our study provides important preliminary data for
the potential utilization of minoxidil as an adjuvant
pharmacological treatment for reducing fibrosis in the
clubfoot medial side after local administration. This
treatment could potentially reduce tissue contracture
in relapsed patients and thus accelerate and/or improve
the outcomes of current conservative methods, espe­
cially in cases when the relapsed patients are not ade­
quately responsive to Ponseti treatment. There is
ongoing research on antifibrotic drugs for use in var­
ious fibrotic diseases (especially pulmonary and liver
fibrosis), but to the best of our knowledge little or no
attention is being paid to their similar potential for
treating clubfoot. Further investigation is needed with
a view to harnessing this potential.
Acknowledgments
We would also like to express our thanks to Daniel
Hadraba, Ph.D. for consultations on statistical methods, to
Marta Vandrovcova, Ph.D. for proofreading the article, and
to the personnel of Bulovka Hospital for their cooperation
in sample acquisition and storage. Mr. Robin Healey is
gratefully acknowledged for his language revision of the
manuscript.
Disclosure statement
The authors report no conflict of interest.
Funding
This work was supported by the Ministry of Health of the
Czech Republic [AZV 17-31564A] and by Charles University
[project GA UK No. 336218]. Some of the images used in this
study were obtained from the confocal microscope unit,
EFFECT OF MINOXIDIL ON CLUBFOOT-DERIVED CELLS 567
which is supported by the Ministry of Education, Youth
and Sports of the Czech Republic [LM2015062 Czech-
BioImaging] and by the European Regional Development
Fund [project OPPK BrainView CZ.2.16/3.1.00/21544].
Author contributions
J.K.: Conceptualization, Methodology, Investigation, Validation,
Project administration, Writing – Original draft. M.D.:
Conceptualization, Investigation, Validation, Formal analysis,
Visualization, Funding acquisition, Writing – Original draft. M.
P.: Conceptualization, Investigation. D.V.: Validation, Formal
analysis. A.E.: Conceptualization, Supervision, Funding acquisi­
tion. M.O.: Resources, Writing – Review. J.M.: Investigation,
Writing – Review. L.B.: Supervision, Writing – Review and
Editing. T.N.: Supervision, Writing – Review and Editing.
ORCID
Jarmila Knitlova http://orcid.org/0000-0003-4607-4497
Martina Doubkova http://orcid.org/0000-0002-4955-4093
Martin Plencner http://orcid.org/0000-0002-9540-9188
David Vondrasek http://orcid.org/0000-0001-6432-1012
Adam Eckhardt http://orcid.org/0000-0003-4757-5226
Martin Ostadal http://orcid.org/0000-0002-3282-9847
Jana Musilkova http://orcid.org/0000-0001-9605-9821
Lucie Bacakova http://orcid.org/0000-0002-1818-9484
Tomas Novotny http://orcid.org/0000-0002-3855-0038
References
1. Ponseti IV, Smoley EN.Congenital Club Foot: the
Results of Treatment. JBJS. 1963;45(2):261.
2. Dimeglio A, Canavese F. The French functional physi­
cal therapy method for the treatment of congenital
clubfoot. J Pediatr Orthop B. 2012;21(1):28–39.
doi:10.1097/BPB.0b013e32834ee5f8
3. Dobbs MB, Gurnett CA. Update on Clubfoot: etiology
and Treatment. Clin Orthop Relat Res. 2009;467
(5):1146–1153. doi:10.1007/s11999-009-0734-9
4. The RC. Ponseti method for the treatment of congeni­
tal club foot: review of the current literature and treat­
ment recommendations. Int Orthop. 2013;37(9):1747–
1753. doi:10.1007/s00264-013-2031-1
5. Hosseinzadeh P, Kiebzak G, Dolan L, Zionts L,
Morcuende J. Management of Clubfoot Relapses With
the Ponseti Method: results of a Survey of the POSNA
Members. PubMed - NCBI. 2019;39(1):38–41.
doi:10.1097/BPO.0000000000000953
6. Ošťádal M, Chomiak J, Dungl P, Frydrychová M,
Burian M. Comparison of the short-term and long-
term results of the Ponseti method in the treatment
of idiopathic pes equinovarus. Int Orthop. 2013;37
(9):1821–1825. doi:10.1007/s00264-013-2033-z
7. Jeans KA, Karol LA, Erdman AL, Stevens WR.
Functional Outcomes Following Treatment for
Clubfoot: ten-Year Follow-up. J Bone Joint Surg.
2018;100(23):2015–2023. doi:10.2106/JBJS.18.00317
8. Dunn PM.Congenital postural deformities: perinatal
associations. Proc R Soc Med. 1972;65(8):735–738.
568 J. KNITLOVA ET AL.
9. Robertson WW, Corbett D. Congenital clubfoot. Month
of conception. Clin Orthop Relat Res. 1997;338:14–18.
doi:10.1097/00003086-199705000-00004.
10. Bonnell J, Cruess R.Anomalous Insertion of the Soleus
Muscle as a Cause of Fixed Equinus Deformity: A Case
Report. J Bone Joint Surg. 1969;51(5):999–1000.
11. Hootnick DR, Levinsohn EM, Crider RJ, Packard DS.
Congenital arterial malformations associated with club­
foot. A report of two cases. Clin Orthop Relat Res.
1982;167:160–163.
12. Sadler BA, Gurnett CB, Dobbs M. The genetics of
isolated and syndromic clubfoot. J Children’s Orthop.
2019;13:238–244. doi:10.1302/1863-2548.13.190063.
13. Fukuhara K, Schollmeier G, Uhthoff HK. The patho­
genesis of club foot. A histomorphometric and immu­
nohistochemical study of fetuses. J Bone Joint Surg.
British Volume. 1994;76(3):450–457.
14. Ippolito E, Farsetti P, Caterini R, Tudisco C. Long-
term comparative results in patients with congenital
clubfoot treated with two different protocols. J Bone
Joint Surg. American Volume. 2003;85(7):1286–1294.
doi:10.2106/00004623-200307000-00015.
15. Zimny ML, Willig SJ, Roberts JM, D’Ambrosia RD. An
electron microscopic study of the fascia from the medial
and lateral sides of clubfoot. J Pediatr Orthop. 1985;5
(5):577–581. doi:10.1097/01241398-198509000-00014
16. Eckhardt A, Novotny T, Doubkova M, Hronkova L,
Vajner L, Pataridis S, Hadraba D, Kulhava L, Plencner
M, Knitlova J, et al. Novel contribution to clubfoot patho­
genesis: the possible role of extracellular matrix proteins. J
Orthop Res. 2019;37(3):769–778. doi:10.1002/jor.24211.
17. Li C, Nguyen Q, Cole WG, Alman BA.Potential treat­
ment for clubfeet based on growth factor blockade. J
Pediatr Orthop. 2001;21(3):372–377.
18. Ošt’ádal M, Eckhardt A, Herget J, Mikšík I, Dungl P,
Chomiak J, Frydrychová M, Burian M. Proteomic ana­
lysis of the extracellular matrix in idiopathic pes equi­
novarus. Mol Cell Biochem. 2015;401(1–2):133–139.
doi:10.1007/s11010-014-2300-3
19. Kerling A, Stoltenburg-Didinger G, Grams L, Tegtbur
U, Horstmann H, Kück M, Mellerowicz H. The con­
genital clubfoot - immunohistological analysis of the
extracellular matrix. Orthop Res Rev. 2018;10:55–62.
doi:10.2147/ORR.S156449.
20. Hersh A. The role of surgery in the treatment of club feet. J
Bone Joint Surg. American Volume. 1967;49(8):1684–
1696.
21. Branton MH, Kopp JBTGF-Β. and fibrosis. Microbes
Infect. 1999;1(15):1349–1365. doi:10.1016/S1286-4579
(99)00250-6
22. McVicker BL, Bennett RG. Novel Anti-fibrotic
Therapies. Front Pharmacol. 2017;8:318. doi:10.3389/
fphar.2017.00318.
23. Collagenase DI. Treatment in Dupuytren Contractures:
A Review of the Current State Versus Future Needs.
Rheumatol Ther. 2016;3(1):43–51. doi:10.1007/s40744-
016-0027-1
24. Poon R, Li C, Alman BA. Beta-catenin Mediates Soft
Tissue Contracture in Clubfoot. Clin Orthop Relat Res.
2009;467(5):1180–1185. doi:10.1007/s11999-008-0692-7
25. Food and Drug Administration. 2017 First Generic
Drug Approvals; 2017 [accessed 2020 Feb 6]. https://
www.fda.gov/drugs/first-generic-drug-approvals/2017-
first-generic-drug-approvals
26. Clinic M. Minoxidil (Topical Route) Side Effects; 2020
[accessed 2020 Feb 6]. https://www.mayoclinic.org/
drugs-supplements/minoxidil-topical-route/side-
effects/drg-20068750?p=1
27. RxList. Minoxidil (Minoxidil Tablets): uses, Dosage,
Side Effects, Interactions, Warning; 2018 Oct 29
[accessed 2020 Feb 6]. https://www.rxlist.com/minoxi
dil-drug.htm
28. Hautala T, Heikkinen J, Kivirikko KI, Myllylä R.
Minoxidil specifically decreases the expression of lysine
hydroxylase in cultured human skin fibroblasts.
Biochem J. 1992;283(Pt 1):51–54.
29. Murad S, Pinnell SR.Suppression of fibroblast prolif­
eration and lysyl hydroxylase activity by minoxidil. J
Biol Chem. 1987;262(25):11973–11978.
30. Piersma B, Bank RA. Collagen cross-linking mediated
by lysyl hydroxylase 2: an enzymatic battlefield to
combat fibrosis. Essays Biochem. 2019;63(3):377–387.
doi:10.1042/EBC20180051
31. Bailey AJ, Bazin S, Sims TJ, Le Lous M, Nicoletis C,
Delaunay A. Characterization of the collagen of human
hypertrophic and normal scars. Biochimica et
Biophysica Acta (BBA) - Protein Struct. 1975;405
(2):412–421. doi:10.1016/0005-2795(75)90106-3
32. Moriguchi T, Fujimoto D. Crosslink of Collagen in
Hypertrophic Scar. J Invest Dermatol. 1979;72(3):143–
145. doi:10.1111/1523-1747.ep12530609
33. van der Slot AJ, Zuurmond A-M, van den Bogaerdt AJ,
Ulrich MMW, Middelkoop E, Boers W, Karel RH,
DeGroot J, Huizinga TWJ, Bank RA. Increased forma­
tion of pyridinoline cross-links due to higher telopep­
tide lysyl hydroxylase levels is a general fibrotic
phenomenon. Matrix Biol. 2004;23(4):251–257.
doi:10.1016/j.matbio.2004.06.001
34. Keeley E, Mehrad B, Strieter R. The role of circulating
mesenchymal progenitor cells (fibrocytes) in the patho­
genesis of fibrotic disorders. Thromb Haemost.
2009;101(4):613–618. doi:10.1160/TH08-11-0726
35. Hinz B, Phan SH, Thannickal VJ, Galli A, Bochaton-Piallat
M-L, Gabbiani G. The Myofibroblast. Am J Pathol.
2007;170(6):1807–1816. doi:10.2353/ajpath.2007.070112
36. Sidney LE, Branch MJ, Dunphy SE, Dua HS, Concise
Review: HA. Evidence for CD34 as a Common Marker
for Diverse Progenitors: CD34 as a Common Marker
for Diverse Progenitors. STEM CELLS. 2014;32
(6):1380–1389. doi:10.1002/stem.1661
37. Peng H, Herzog EL. Fibrocytes: emerging effector cells
in chronic inflammation. Curr Opin Pharmacol.
2012;12(4):491–496. doi:10.1016/j.coph.2012.03.002
38. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V,
Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld
S, Schmid B, et al. Fiji: an open-source platform for
biological-image analysis. Nat Methods. 2012;9(7):676–
682. doi:10.1038/nmeth.2019.
39. International Organization for Standardization. ISO
10993-5:2009(E): Biological evaluation of medical
devices - Part 5: Tests for in vitro cytotoxicity; 2009.
40. Pinnell SR, Murad S.Effects of minoxidil on cultured
human skin fibroblasts. Dermatologica. 1987;175(Suppl
2):12–18.

41. Priestley GC, Lord R, Stavropoulos P. The metabolism
of fibroblasts from normal and fibrotic skin is inhibited
by minoxidil in vitro. Br J Dermatol. 1991;125(3):217–
221. doi:10.1111/j.1365-2133.1991.tb14743.x
42. Zuurmond A, Vanderslotverhoeven A, Vandura E,
Degroot J, Bank R. Minoxidil exerts different inhibitory
effects on gene expression of lysyl hydroxylase 1, 2, and
3: implications for collagen cross-linking and treatment
of fibrosis. Matrix Biol. 2005;24(4):261–270.
doi:10.1016/j.matbio.2005.04.002
43. Shao S, Zhang X, Duan L, Fang H, Rao S, Liu W, Guo B,
Zhang X. Lysyl Hydroxylase Inhibition by Minoxidil
Blocks Collagen Deposition and Prevents Pulmonary
Fibrosis via TGF-β1/Smad3 Signaling Pathway. Med Sci
Monit. 2018;24:8592–8601. doi:10.12659/MSM.910761.
44. Alvarado DM, McCall K, Aferol H, Silva MJ, Garbow
JR, Spees WM, Patel T, Siegel M, Dobbs MB, Gurnett
CA. Pitx1 haploinsufficiency causes clubfoot in
humans and a clubfoot-like phenotype in mice. Hum
Mol Genet. 2011;20(20):3943–3952. doi:10.1093/hmg/
ddr313
45. Collinson JM, Lindström NO, Neves C, Wallace K,
Meharg C, Charles RH, Ross ZK, Fraser AM, Mbogo I,
Oras K, et al. The developmental and genetic basis of
‘clubfoot’ in the peroneal muscular atrophy mutant
mouse. Development (Cambridge, England). 2018;145:3.
46. Kakkar A, Traverso G, Farokhzad OC, Weissleder R,
Langer R. Evolution of macromolecular complexity in
EFFECT OF MINOXIDIL ON CLUBFOOT-DERIVED CELLS 569
drug delivery systems. Nature Rev Chem. 2017;1
(8):0063. doi:10.1038/s41570-017-0063
47. Wynn TA. Common and unique mechanisms regulate
fibrosis in various fibroproliferative diseases. J Clin
Invest. 2007;117(3):524–529. doi:10.1172/JCI31487
48. Chen CZ, Raghunath M. Focus on collagen: in vitro
systems to study fibrogenesis and antifibrosis state of
the art. Fibrogenesis Tissue Repair. 2009;2:7.
doi:10.1186/1755-1536-2-7.
49. Devkota AK, Veloria JR, Guo HF, Kurie JM, Cho EJ,
Dalby KN. Development of a High-Throughput Lysyl
Hydroxylase (LH) Assay and Identification of Small-
Molecule Inhibitors against LH2. SLAS Discovery.
2019;24(4):484–491. doi:10.1177/2472555218817057
50. Wang C, Luosujärvi H, Heikkinen J, Risteli M, Uitto L,
Myllylä R. The third activity for lysyl hydroxylase 3:
galactosylation of hydroxylysyl residues in collagens in
vitro. Matrix Biol. 2002;21(7):559–566. doi:10.1016/
s0945-053x(02)00071-9
51. Merl-Pham J, Basak T, Knüppel L, Ramanujam D,
Athanason M, Behr J, Engelhardt S, Eickelberg O,
Hauck SM, Vanacore R, et al. Quantitative proteomic
profiling of extracellular matrix and site-specific col­
lagen post-translational modifications in an in vitro
model of lung fibrosis. Matrix Biol Plus. 2019;1:100005.
52. Dallon JC, Ehrlich HP. A review of fibroblast-populated
collagen lattices. Wound Repair Regenerat. 2008;16
(4):472–479. doi:10.1111/j.1524-475X.2008.00392.x