Mammalian Cell Behavior On Hydrophobic Substrates Influence of Surface Properties
Mammalian Cell Behavior On Hydrophobic Substrates Influence of Surface Properties
Mammalian Cell Behavior On Hydrophobic Substrates Influence of Surface Properties
and interfaces
Review
Mammalian Cell Behavior on Hydrophobic
Substrates: Influence of Surface Properties
Michele Ferrari 1,2, * , Francesca Cirisano 1 and M. Carmen Morán 2,3, *
1 CNR-ICMATE Istituto di Chimica della Materia Condensata e di Tecnologie per l’Energia, via De Marini, 6,
16149 Genova, Italy; [email protected]
2 Institut de Nanociència i Nanotecnologia—IN2 UB, Universitat de Barcelona, Avda. Diagonal, 645,
08028 Barcelona, Spain
3 Departament de Bioquímica i Fisiologia, Secció de Fisiologia—Facultat de Farmàcia i Ciències de
l’Alimentació, Universitat de Barcelona, Avda. Joan XXIII s/n, 08028 Barcelona, Spain
* Correspondence: [email protected] (M.F.); [email protected] (M.C.M.)
Received: 26 March 2019; Accepted: 7 May 2019; Published: 7 May 2019
Abstract: The influence of different surface properties holding to a modification of the substrate
towards hydrophobic or superhydrophobic behavior was reviewed in this paper. Cell adhesion, their
communication, and proliferation can be strongly manipulated, acting on interfacial relationship
involving stiffness, surface charge, surface chemistry, roughness, or wettability. All these features
can play mutual roles in determining the final properties of biomedical applications ranging from
fabrics to cell biology devices. The focus of this work is the mammalian cell viability in contact with
moderate to highly water repellent coatings or materials and also in combination with hydrophilic
areas for more specific application. Few case studies illustrate a range of examples in which these
surface properties and design can be fruitfully matched to the specific aim.
1. Introduction
Cell adhesion between cells and surfaces is a crucial controlling parameter on biological processes
ranging from activation, differentiation, migration, proliferation, or purely survival [1]. These adhesive
interactions between cells and biomaterials could determine the final applicability of implanted
prostheses by regulation of the inflammatory responses and degree of connection to immediate tissues.
Nowadays, it is widely recognized that the control of biological adhesion is controlled by specific
interactions between cell surface receptors and their ligands, although the influence of nonspecific
forces must not be forgotten [2].
Changes in cell adhesion have been detected in a wide range of diseases such as arthritis [3,4],
cancer [5–7], osteoporosis [8,9], and atherosclerosis [10,11]. In general, cell adhesiveness is reduced
in human cancers, where the diminished intercellular adhesiveness allows cancer cells to migrate,
resulting in the loss of the histological structure, which is the morphological distinctive of malignant
tumors [7]. Tumor cells are characterized by changes in adhesion to extracellular matrix (ECM), which
may be related to their invasive and metastatic potential. Cell–matrix as well as cell–cell interactions
have been reported to be cell type- and oncogene-specific [12–14].
When cells adhere to the surface of a material, a sequence of physicochemical reactions between
the cells and the material interface occurs. The first event after the biomaterial is implanted into an
organism is the protein adsorption to its surface, which mediates the cell adhesion and offers signals to
the cell through the cell adhesion receptors, mainly of the integrin type [15]. Cells can adhere on the
the surfaces and release active compounds, which would mediate deposition of the extra-cellular
surfaces and release active
matrix, differentiation, andcompounds, which would mediate deposition of the extra-cellular matrix,
even cell proliferation.
differentiation, and even cell proliferation.
Interfacial interactions between cell/tissue and surfaces correlate quite well with many
Interfacial
important phenomenainteractions betweensystems
in biological cell/tissueand and surfaces
have correlate quite
been considered well
for the with many important
development of several
phenomena in biological systems and have been considered for the
artificial biomaterials and applications [15–18]. To fabricate scaffolds and implants development of severalfor artificial
tissue
biomaterials and applications [15–18]. To fabricate scaffolds and implants
engineering and regenerative medicine, biomimetic materials promoting a favorable response from for tissue engineering and
regenerative
cells and tissuesmedicine, biomimetic
are required. Most materials
biomedical promoting
devices,a however,
favorable response
because of from
thecells andwith
contact tissues are
blood
required. Most biomedical devices, however, because of the contact with blood
or tissues, require those bioinert polymers suppressing nonspecific adhesions that could induce or tissues, require those
bioinert
trombosis polymers suppressing nonspecific
and immunological adhesions that could induce trombosis and immunological
responses [19–24].
responses [19–24].
The biocompatibility of a material refers to its capability to provide appropriate support to
Theactivity,
cellular biocompatibility
includingofthe a material
stimulation refers to its capability
of molecular to provideprocesses,
and mechanical appropriate support
which would to
cellular
optimizeactivity, including thewithout
tissue regeneration, stimulation of molecular
producing and mechanical
any undesirable response processes, which would
in the eventual host,
optimize tissue regeneration, without producing any undesirable response
either at local or systemic level [25–27]. When the in vitro biocompatibility of a biomaterial in the eventual host, either is
at local or systemic level [25–27]. When the in vitro biocompatibility of a biomaterial
determined, it depends mainly on its own surface properties, such as stiffness, surface charge, is determined, it
depends
chemicalmainly on its ownroughness,
functionalities, surface properties, such as stiffness,
and wettability, and on the surface charge, chemical
cell interactions withfunctionalities,
the scaffold’s
roughness, and wettability, and on the cell interactions with the scaffold’s
degradation products [28]. Besides this, the culture medium and conditions, as well as degradation products
the type [28].
of
Besides this, the culture medium and conditions, as well as the type of cell,
cell, are important parameters that also modulate the behaviour of cells. Figure 1 summarizes theare important parameters
that
mainalso modulate
surface the behaviour
properties that may of cells.
affectFigure 1 summarizes
cell behavior the main
in terms surface
of the properties
adhesion, that may
morphology,
affect cell behavior in terms of the adhesion,
metabolism, and proliferation, among others. morphology, metabolism, and proliferation, among others.
Figure 1.1.Schematic
Figure Schematicrepresentation of the
representation mainmain
of the surface properties
surface and their
properties anddirect
theireffect oneffect
direct cell behavior.
on cell
behavior.
Surfaces prepared with particular characteristics can control adhesion and proliferation of cells
on substrate
Surfacessurfaces.
preparedDifferent techniques
with particular have been applied,
characteristics aimed
can control at introducing
adhesion chemical groups
and proliferation of cells
addressing the behavior to hydrophilicity or hydrophobicity, as well as physically
on substrate surfaces. Different techniques have been applied, aimed at introducing chemical groups modifying the
surface
addressing the behavior to hydrophilicity or hydrophobicity, as well as physically modifying the
in terms of roughness, surface energy, or morphology. Plasma treatment is among one of the
most popular and reliable methods for an appropriate surface modification [29–32].
surface in terms of roughness, surface energy, or morphology. Plasma treatment is among one of the
mostAs the prediction
popular of a methods
and reliable precise mechanism of interaction
for an appropriate of modification
surface a surface in biological
[29–32]. environment is
complicated by the composition of body fluids (water, various proteins), as
As the prediction of a precise mechanism of interaction of a surface in biological well as by cells’ synergistic
environment is
interaction [33,34], surface properties in terms of surface wettability with respect
complicated by the composition of body fluids (water, various proteins), as well as by cells’ to their hydrophobicity
or hydrophilicity
synergistic have [33,34],
interaction an important
surfacerole in influencing
properties in termstheofbiological response of
surface wettability witha biomaterial [35].
respect to their
In this work, we review
hydrophobicity the available
or hydrophilicity literature
have about a few
an important rolephysico-chemical
in influencing the and mechanical
biological properties
response of a
of a surface and dynamic processes at the interfaces significantly affecting the behavior
biomaterial [35]. In this work, we review the available literature about a few physico-chemical and of mammalian
cells, together
mechanical with caseofstudies
properties focused
a surface on highly
and dynamic repellentatsubstrates.
processes the interfaces significantly affecting the
behavior of mammalian cells, together with case studies focused on highly repellent substrates.
Colloids Interfaces 2019, 3, 48 3 of 16
analysis, a two-layer material model composed of one thin and stiff plasma-oxidized layer on a
Colloids Interfaces 2019, 3, 48 4 of 16
thicker layer of more elastic bulk polymer could fit the experimental data. This study clearly
described a novel approach for characterizing the effect of the biopolymer surface modifications on
material modelmechanics
cell–biomaterial composed of one thin and stiff plasma-oxidized layer on a thicker layer of more elastic
bulk polymer could fit the experimental data. This study clearly described a novel approach for
2.2. Surface Charge
characterizing and
the Chemical
effect of theFunctionalities
biopolymer surface modifications on cell–biomaterial mechanics
It has long been known by electrophoretic measurements that mammalian cells bear a net
2.2. Surface Charge and Chemical Functionalities
negative charge [44]. Consequently, the interaction between cells and negative surfaces can be used
It has cell
to minimize longadhesion
been known by electrophoretic
by electrostatic repulsion.measurements
The results usingthatlow
mammalian cells medium,
ionic strength bear a net
negative
such charge [44].media
as physiological Consequently, the interaction
[45] and grafted polymerbetween
brushescells and negative
on substrates [46],surfaces can be used to
have demonstrated
minimize cell adhesion by electrostatic repulsion. The results using low ionic
that cell–surface adhesion could be prevented. When the protein adsorption on negatively charged strength medium, such
as physiological
surfaces media
is considered, the[45] and grafted
degree polymer
of adsorption brushes
would on substrates
depend [46],strength
on the ionic have demonstrated
of the nearby that
cell–surface adhesion could be prevented. When the protein adsorption on negatively
solution [47], mainly mediated by the release of the corresponding counterion of the polymers into charged surfaces
is solution.
the considered, the degree of adsorption would depend on the ionic strength of the nearby solution [47],
mainly mediated
The effect by the charge
of surface release of
hasthe corresponding
been extensively counterion
analyzed inofthethecell
polymers into the
attachment solution.
mechanism.
The effect of surface charge has been extensively analyzed in the cell attachment
The amount of positive charges on the surfaces can strongly influence cell behavior. Several studies mechanism.
Thedemonstrated
have amount of positive
how cellcharges on the
adhesion andsurfaces can strongly
proliferation influence cell
can be modulated bybehavior. Several
surface charge studies
density.
have 2demonstrated
Figure demonstrateshow how, cell
asadhesion
the degreeandofproliferation
charge densitycanof
behydrogels
modulated by surface
based charge density.
on 2-hydroxyethyl
Figure 2 demonstrates how, as the degree of charge density of
methacrylate (HEMA) and 2-methacryloxyethyl trimethyl ammonium chloride (MAETAC)hydrogels based on 2-hydroxyethyl
methacrylate
copolymer (HEMA)
increased, celland 2-methacryloxyethyl
adhesion trimethyl
and proliferation ammonium
are favored [48]. chloride (MAETAC) copolymer
increased, cell adhesion and proliferation are favored [48].
surfaces with amine and hydroxyl groups confirmed alkaline phosphatase enzymatic activity and
matrix mineralization, and up-regulated osteoblast-specific gene expression in comparison with that
observed with surfaces functionalized with alkyl and carboxyl groups [56].
Surface charge, along with wettability properties, has been extensively reported in the literature
as two physical issues governing protein adsorption and cell adhesion. Although the comparison
between these two driving forces in terms of their independent and cooperative effects on cell adhesion
is rarely explored, a recent work has developed a protocol featuring two-dimensional control over both
surface charge and wettability influence [57]. This approach allows the control of both the assembly of
the polyion charge density in the layer-by-layer (LbL) and the polyion side-chain chemical structures.
Surface isoelectric points ranging from 5 to 9 and water contact angles from 35◦ to 70◦ on the 2D
property matrix were compared. The relationship between the two surface parameters influences both
protein (bovine serum albumin, lysozyme) adsorption and 3T3 fibroblast cell adhesion. The highest
cell adhesion was found by the combined effects of positive charge and hydrophilicity, while a most
limited cell adhesion was promoted by negative charge and hydrophobicity.
influencing surface energy and controlling wettability by covering the structures with suitable coatings,
acting on the surface chemistry without affecting morphology. Lower roughness ratios indicated
better fibroblast cell adhesion, non-monotonically dependent on surface energy, but independent
from surface wettability and chemistry. The role of size and shape of surface topography on cell
attachment is well underlined, showing how laser structured scaffolds, at controlled roughness ratio
and surface chemistry, could be a promising in vivo method to define 3D cell–biomaterials interactions.
The main original finding emerging from this paper is the synergistic role of the roughness degree
and surface chemistry defining wettability or surface energy features of the substrate. These two
parameters are in turn responsible for switching properties from cell-phobic to cell-philic behavior or
Colloids Interfaces 2019, 3, 48
from super-hydrophobic 6 of 16
to super-hydrophilic with a well-defined transition point in the wettability.
Figure 3.
Figure Scanningelelctron
3. Scanning elelctronmicroscopy
microscopyimages
imagesofofMG63
MG63cells
cellsattached
attachedon onthe
thepolycarbonate
polycarbonate(PC)
(PC)
membrane surfaces with micropore sizes ranging from 0.2 (A), 0.4 (B) 1 (C), 3 (D), 5 (E), and
membrane surfaces with micropore sizes ranging from 0.2 (A), 0.4 (B) 1 (C), 3 (D), 5 (E), and 8 mm 8 mm (F).
Reprinted from reference [60] with permission from Elsevier.
(F). Reprinted from reference [60] with permission from Elsevier.
The effect of both chemical composition and topography on the surface have been evidenced
The adhesion and viability of fibroblast cells on highly rough three-dimensional (3D) silicon (Si)
by the introduction of hydrophilic Pluronic copolymer onto the polyurethane (PU) surface, with a
surfaces have been studied in the work of [66]. The aim was to investigate culture surfaces with
lotus leaf-like
gradient topography
roughness ratios and[67]. The obtained
wettabilities surfacesbywere
produced demonstrated
femtosecond to beStructuring
(fs) laser. inert to both protein
Si wafers
adsorption and L929 fibroblasts cells adhesion. Recent studies have pointed
resulted in controlled dual-scale roughness composed by conical spikes at both the micro- and the out the influence of
wettability instead
nano-scale. of polymer
By changing the topography
laser pulse on the inhibition
fluency, differentof cell adhesionlevels
roughness on flatcould
and rough surfaces.
be obtained,
influencing
3. Dynamicsurface
Processes energy
at theand controlling wettability by covering the structures with suitable
Interfaces
coatings, acting on the surface chemistry without affecting morphology. Lower roughness ratios
Surface
indicated tension
better related
fibroblast cellphenomena like wetting and wettability
adhesion, non-monotonically dependent are dynamicenergy,
on surface interfacial
but
processes and
independent critical
from surface factors for the
wettability andcell behavior.TheFor
chemistry. roleinstance,
of size and biological relevanttopography
shape of surface properties
likecell
on proteins’ conformation
attachment is well are strongly affected
underlined, showingbyhow interfacial adsorptionscaffolds,
laser structured and adsorbed material,
at controlled
roughness ratio and surface chemistry, could be a promising in vivo method to define 3D based
with an indirect influence on the interactions between cell and substrate [68–71]. Research cell–
on polydimethylsiloxane (PDMS) substrate confirmed that this is an easily
biomaterials interactions. The main original finding emerging from this paper is the synergistic role tunable material for
cell–substrate interactions studies because of its mechanical properties influencing
of the roughness degree and surface chemistry defining wettability or surface energy features of the surface chemistry
and cell response
substrate. These two [72]. Changesare
parameters in in
formulation resulted
turn responsible forinswitching
significant changesfrom
properties in elastic modulus
cell-phobic to
without changing the wettability, which shows an inverse behavior with stiffness
cell-philic behavior or from super-hydrophobic to super-hydrophilic with a well-defined transition after polyelectrolytes’
adsorption.
point Under these different composition conditions, cell attachment and spreading are governed
in the wettability.
by surface properties
The effect of bothatchemical
early stages, while cell and
composition growth depends on
topography on mechanical
the surface properties
have beenat longer times.
evidenced by
Under another perspective, applications requiring a particular cell response
the introduction of hydrophilic Pluronic copolymer onto the polyurethane (PU) surface, with a lotus on a specific substrate
can benefit
leaf-like of a surface
topography [67].modification,
The obtainedinducing
surfacesselective adhesion of to
were demonstrated cells
be through
inert to aboth
competitive
protein
adsorption and
adsorption [28]. L929
In recent years, more
fibroblasts cells and more attention
adhesion. has been
Recent studies paidpointed
have to extreme
out wettability
the influencestates,
of
wettability instead of polymer topography on the inhibition of cell adhesion on flat and rough
surfaces.
adsorption. Under these different composition conditions, cell attachment and spreading are
governed by surface properties at early stages, while cell growth depends on mechanical properties
atInterfaces
Colloids longer times.
2019, 3, 48 7 of 16
Under another perspective, applications requiring a particular cell response on a specific
substrate can benefit of a surface modification, inducing selective adhesion of cells through a
which can address
competitive the ability
adsorption [28].ofInany solidyears,
recent surface to and
more be wetted when inhas
more attention contact
been with
paid atoliquid
extreme to
discriminate biological environments. Water contact angle (WCA), although not
wettability states, which can address the ability of any solid surface to be wetted when in contact exclusively, is still the
key experimental
with a liquidtool to assess such
to discriminate particular
biological characteristics;
environments. thus,contact
Water according angleto the valuealthough
(WCA), of the angle,
not
a surface is considered
exclusively, hydrophobic
is still the key experimentalwhentool
it isto
between 150◦particular
assess such and 90◦ , characteristics;
but superhydrophobic when
thus, according
WCAtoisthe valuethan
higher of the ◦ , hydrophilic
150angle, a surfacewhen
is considered 90◦ and 10
hydrophobic
it is between ◦ , and
when it issuperhydrophilic
between 150° and 90°, but
when it is
than 10 . ◦
lowersuperhydrophobic when WCA is higher than 150°, hydrophilic when it is between 90° and 10°, and
superhydrophilic
With the WCA being when it is
the lower
angle than 10°. by the droplet of water and the surface, the wettability
subtended
is derived from an inverse measure orsubtended
With the WCA being the angle by the
interpretation ofdroplet
the WCA of water
and, and the surface,
in addition, its the wettability
measurement
is derived from an inverse measure or interpretation of the WCA and,
requires different experimental and theoretical tools when approaching heterogeneous substrates orin addition, its measurement
requires
non-ideal differentHistorically,
materials. experimental and surface
some theoretical toolshave
models whenbeen
approaching
proposedheterogeneous
to predict andsubstrates
explain the or
way non-ideal materials. Historically, some surface models have been proposed to predict and explain the
surface interacts with water [73]. For instance, the Young model [74] is applicable for smooth
way surface interacts with water [73]. For instance, the Young model [74] is applicable for smooth
and chemically homogeneous surfaces, while those who mostly contributed to the interpretation of
and chemically homogeneous surfaces, while those who mostly contributed to the interpretation of
such extreme wettability states like artificial superhydrophobic surfaces are the Wenzel [75] and the
such extreme wettability states like artificial superhydrophobic surfaces are the Wenzel [75] and the
Cassie–Baxter models [76] (Figure 4). Both starting from the observation of animal water repellent
Cassie–Baxter models [76] (Figure 4). Both starting from the observation of animal water repellent
surfaces, Wenzel considers that the droplet can make full contact with the surface, and, by contrast,
surfaces, Wenzel considers that the droplet can make full contact with the surface, and, by contrast,
Cassie and and
Cassie Baxter better
Baxter betterunderstood
understood thethe
superhydrophobicity,
superhydrophobicity,taking
takingintointoaccount
accountthe
the air
air entrapped
entrapped
under the droplet between the surface projections.
under the droplet between the surface projections.
Figure 4. Representation
Figure of the
4. Representation Young
of the (a),(a),
Young Wenzel (b)(b)
Wenzel and Cassie-Baxter
and Cassie-Baxter(c)
(c)models
modelsshowing
showing the
the main
main
differences in terms of surface and interactions with the droplet.
differences in terms of surface and interactions with the droplet.
Additionally, the control of protein adsorption, either through the inhibition of entire or selective
adsorption to the biomaterial surface, has important implications for many applications discussed in
this review, from cellular interactions to diagnostic and drug delivery platforms. Hydrophobic surfaces
have been traditionally considered as suppressors of protein adsorption, with their hydrophobic
residues within the folded three-dimensional structure. Recently, this approach has been clarified,
showing that proteins adsorb to hydrophobic materials by direct interactions of hydrophobic patches
on the protein surface, as well as through denaturation processes, allowing the protein to bind to the
material surface, exposing the internal hydrophobic residues.
On the other hand, minimal protein denaturation or unfolding is generally found because of the
interaction of the hydrophilic moieties on the proteins surface with a hydrophilic biomaterial surface.
Nevertheless, the development of a biomaterial or a biomedical application has to take into account
protein adsorption phenomena of a large number of macromolecules in vivo with different structural
and physico-chemical properties [86–88]. Recently, mixed superhydrophobic–superhydrophilic
patterned surfaces could exploit such opposite properties to precisely organize and cultivate closely
adjacent arrays of different cell lines, for diagnostics and cell signaling studies or tissue engineering [89].
4. Case Studies
The development of biomedical applications seeks biomaterials with a suitable anti-adhesive effect
on cells or tissues in the body and, at the same time, with health and body protection from traumas
or foreign bodies, with tissue adhesion barriers physically isolating wounds or devices [90]. In the
following section, a few selected applications describe the behaviour of mammalian cells on surfaces
coated by low wettable systems, ranging from common polymers to more complicate combinations
with opposite behaviours.
a. The influence of the composition of poly(dimethylsiloxane) (PDMS) was investigated, studying
the attachment and growth properties of several different types of mammalian cells: primary
human umbilical artery endothelial cells (HUAECs), transformed 3T3 fibroblasts (3T3s),
transformed osteoblast-like cells (MC3T3-E1), and transformed epithelial cells (HeLa). Cells’
growth has been studied on PDMS at different ratios of curing agent, that is, 10:1 v/v (normal
PDMS, PDMSN), 10:3 v/v (PDMSCA), and 10:0.5 v/v (PDMSB), as well as on extracted PDMS
(normal PDMS with reduced quantities of low molecular-weight oligomers, PDMSN, EX), normal
PDMS extracted and then oxidized (PDMSN, EX, OX). Before the cell attachment step, all surfaces
were exposed to a solution of fibronectin, being fibronectin-coated PDMS as suitable substrate for
culturing mammalian cells [91]. The cell type appeared to be the most influencing factor of cells
compatibility on some surfaces; 3T3 fibroblasts and MC3T3-E1 cells showed detachment from
PDMSN, EX, OX, while HUAECs and HeLa cells detached from the PDMSCA surface. For most
of the cell types on PDMSN, PDMSN, EX, and PDMSB, cell growth was comparable to standard
tissue culture-treated polystyrene (TCPS). Despite Young’s moduli range, the growth rate was
found to be similar for all cells on PDMS substrates and then independent on substrate stiffness.
b. Bioinspired superhydrophobic surfaces were prepared based on intrinsically hydrophobic PDMS
by roughening a structure using surface aggregates of nanoparticles [92]. The wettability data
resulted in a direct dependence of WCA with the increasing of concentration of hydrophobic
TiO2 . A correlation between surface properties and cell–surface interactions supported the
cell adhesion studies carried out in this work. Only the superhydrophobic sample showed a
cell-repellent behavior, with a decreasing of cell viability up to 80% compared with the pure
PDMS film. The surface energy was shown to play a key role in the cell-repellent behavior of the
superhydrophobic sample, because of similarities in the roughness profiles of the two samples.
This work underlines how surface wettability, roughness, and chemistry are parameters of
optimization for developing biomaterial surfaces with controlled cell adhesion behavior.
c. The effect of enhancing the water repellence of the substrate was evaluated by determining the
adhesion and spreading of human fibroblasts on untreated FEP–Teflon (hydrophobized), and
the pure PDMS film. The surface energy was shown to play a key role in the cell-repellent
behavior of the superhydrophobic sample, because of similarities in the roughness profiles of
the two samples. This work underlines how surface wettability, roughness, and chemistry are
parameters of optimization for developing biomaterial surfaces with controlled cell adhesion
behavior.
Colloids Interfaces 2019, 3, 48 9 of 16
c. The effect of enhancing the water repellence of the substrate was evaluated by determining the
adhesion and spreading of human fibroblasts on untreated FEP–Teflon (hydrophobized), and
wasalso
was alsocompared
comparedwith withTCPS
TCPS[93].
[93].Ion
Ionetched
etchedSuperhydrophobic
SuperhydrophobicFEP–Teflon
FEP–Teflon was was prepared
prepared and
and
followed by oxygen glow-discharge, resulting in water contact angles of 140–150 ◦ (untreated
followed by oxygen glow-discharge, resulting in water contact angles of 140°–150° (untreated
FEP–Teflon: 109◦ ). Compared
FEP–Teflon: 109°). Comparedwith withuntreated FEP–Teflon
untreated (209 µm
FEP–Teflon
2
(209 per
μmcell),
2 pera significant decrease
cell), a significant
in the spreading of human skin fibroblasts was observed on superhydrophobic
decrease in the spreading of human skin fibroblasts was observed on superhydrophobic FEP– FEP–Teflon
(158 µm 2
Teflon (158per
μmcell)
2 per (Figure 5). This
cell) (Figure workwork
5). This put in
putevidence thatthat
in evidence adhesion
adhesionandandspreading cancan
spreading be
considered
be consideredtwo twodifferent
differentphenomena;
phenomena;ininfact,
fact,while
whilecell
cellspreading
spreading on on TCPS
TCPS was
was significantly
significantly
higher as compared with FEP–Teflon, the number of adhering cells
higher as compared with FEP–Teflon, the number of adhering cells on TCPS, however, on TCPS, however, was
was
significantlyhigher
significantly higherthanthanononthe
thehydrophobic
hydrophobicFEP–Teflon.
FEP–Teflon.
Figure 5. Light
Light microscopy
microscopy images
images of
of human
human skin
skin fibroblasts attached to tissue culture-treated
polystyrene (TCPS)
polystyrene (TCPS)(a),
(a),untreated
untreated FEP–Teflon
FEP–Teflon (b), hydrophobized
(b), and and hydrophobized FEP–Teflon
FEP–Teflon (c). The(c).
barThe bar
denotes
denotes 37 μm. Reprinted from reference
37 µm. Reprinted from reference [93]. [93].
d. Cell
Cell proliferation
proliferation on ona patterned
a patterned ordered
ordered structure
structure obtained
obtained by plasma by CVD
plasmaandCVD and VUV
VUV irradiation
irradiation as a combination
as a combination of bothhydrophobic
of both highly highly hydrophobic and hydrophilic
and hydrophilic areas wasareas was investigated
investigated in the
inwork
the of
work
[94].ofThe
[94].correlation
The correlation
between between
chemistry,chemistry, physicochemical
physicochemical properties properties of the
of the surface,
surface, and adhesive
and adhesive behaviour behaviour
of cellsofwascellsinvestigated
was investigated
usingusing
such such a surface
a surface as a as a scaffold
scaffold for
for cell
cell culture.
culture. TheThe cellcell selectivity
selectivity for for superhydrophilic
superhydrophilic areas
areas waswas confirmed
confirmed by comparison
by comparison withwith
the
the superhydrophobic
superhydrophobic part part
findingfinding theroughness
the first first roughness
structurestructure
intact. Inintact. In cells
facts, the facts,distributed
the cells
distributed
regularly asregularly as circular
circular arrays alongarrays alongpattern
the surface the surface
with pattern
a distancewith a distance
negative effectnegative effect
over a certain
over
sizea(>400
certainµm)size
on(>400 μm)adhesive
the cell on the cell adhesivewith
extension extension with the neighbours.
the neighbours. Examining Examining
cell behaviourcell
behaviour on superhydrophobic
on superhydrophobic and superhydrophilic
and superhydrophilic surfaces hassurfaces has demonstrated
demonstrated that cells adhered that cells
and
adhered and on
proliferated proliferated
both surfaces;on both
evensurfaces; even on the superhydrophobic
on the superhydrophobic surface,
surface, they divide andthey divide
proliferate
and proliferate
in the presence inoftheconstant
presencecontact.
of constant contact.
In the study,Inthe
therole
study, the roleadsorption
of protein of protein adsorption
in the site
inselectivity
the site selectivity
in the final in the final adhesion
adhesion properties properties on different
on different surfacessurfaces was underlined—far
was underlined—far greater
greater
amounts amounts
of proteins of adsorbed
proteins on adsorbed on the flat
the flat hydrophilic hydrophilic
surface than on thesurface than on the
flat hydrophobic flat
surface.
e. hydrophobic surface.
Cell attachment is governed by differences in surface energy—higher energy hydrophilic surfaces
e. promote adhesion,
Cell attachment while lowby
is governed surface energyinsubstrates
differences usually inhibit cell
surface energy—higher energyadhesion. With
hydrophilic
the aimpromote
surfaces of providing control
adhesion, of low
while cell adhesion,
surface energya combination
substrates of superhydrophobic
usually inhibit cell adhesion.with a
specific high energy component like polydopamine was investigated
With the aim of providing control of cell adhesion, a combination of superhydrophobic with a [95]. Superhydrophobic
surfaceshigh
specific withenergy
their extremely
component lowlike
surface energy can reduce
polydopamine cell adhesion,
was investigated butSuperhydrophobic
[95]. in the presence of
polydopamine,
surfaces with their coatings
extremelycan lowbecome a suitable
surface energysubstrate
can reduceforcell
celladhesion,
adhesion.but In in
other
the words,
presence a
selective polydopamine coating on a cell-repellent superhydrophobic
of polydopamine, coatings can become a suitable substrate for cell adhesion. In other words, abackground can improve
the precision
selective in cell proliferation
polydopamine coating oncontrol systems. superhydrophobic background can improve
a cell-repellent
f. Nonfouling
the precision superhydrophobic
in cell proliferationsilicon control nanowire
systems.(SiNW) substrate in a stable Cassie–Baxter state,
with limited contact with the culture medium, was investigated by exploiting the interface
between nanowires and living cells for applications in fields like biomedical implants, biosensors,
or drug delivery [96]. Vertically aligned SiNW arrays prepared by the stain etching technique
were chemically modified with octadecyltrichlorosilane (OTS), resulting in a superhydrophobic
SiNW surface with a contact angle around 160◦ . Then, by standard optical lithography techniques,
a micropatterned superhydrophilic/superhydrophobic SiNW surface was created for a K1 Chinese
Colloids Interfaces 2019, 3, 48 10 of 16
and structure of the mixed organic–inorganic coating with moderate to high water repellence
can be finely modulated, resulting in controlling the hydrophobicity of the fabric on commercial,
low cost fabric substrates, providing advanced performance. Cell viability on TCPS surfaces
with this superhydrophobic coating has efficiently decreased, independent of the cell line type.
Comparing the ratio values with those observed on uncoated surfaces and a less hydrophobic
coating, the 3T3 or HaCaT cell line decreased their individual responses by 10 times the ratio
values. In case of the HeLa line, the hydrophobic coating of polyester (PES) fabric was very
efficient in minimizing viability in comparison with coating TCPS surfaces. From these results,
tumor cell lines and non-tumor cell lines could be potentially discriminated based on their
adhesion on PES fabrics.
Colloids Interfaces 2019, 3, 48 11 of 16
Figure
Figure 6. 6. Scanning
Scanning electron
electron microscopy
microscopy images of images of attached
HeLa cells HeLa to cells attached
superhydrophobic/to
superhydrophobic/superhydrophilic microtemplates under different experimental conditions:
superhydrophilic microtemplates under different experimental conditions: cells cultured for one, cells
cultured 1
two, andfor one,
four two,
days and(A,C,E)
with four days
and with (A, C,
without E) andair
trapped without
(B,D,F),trapped air (B, Scale
respectively. D, F),bar
respectively.
4 200 mm.
Scale bar ¼from
Reprinted 200 mm. Reprinted
reference frompermission
[97] with reference [97]
fromwith permission from Elsevier.
Elsevier.
5. Conclusions
h. With the aim of improving the correlation of in vitro and in vivo cellular functions, the
requirements
In this work, ofthemimicking
authors havenatural tissuethe
reviewed properties
available(such as chemistry,
literature three-dimensional
outlining the role of surface
structure, mechanical properties, etc) in comparison with traditional polystyrene treated flat
properties involved in the hydrophobic or superhydrophobic state of a surface in relation to mammalian
tissue cell culture dishes for growing, subculturing, and studying cell
cell behaviour including cell adhesion, their communication, and proliferation. Despite behavior are widely
that most
assessed
of these [99]. Interestingly,
properties NIH 3T3
are correctly treated fibroblasts showed
independently significantly
in the literature, fewgreater
of themadhesion and
can together
proliferation on XanoMatrix cell culture dishes; this substrate can be considered a
significantly influence the substrate hydrophobicity, enhancing some aspects of biomedical applications versatile
growth
like fabric platform
durability or with the mimicked
cell detachment. Amongnanoscale geometry
the examples, of natural
a strong tissueon
dependence fibers with true,
the biochemical
tortuous fiber
and biophysical beds. of the cell line and the loss of high water repellence in the case of long time
features
i.exposure to liquids have to characterization
The physico-chemical be evidenced. Nevertheless, the combination
of an alternative with different
platform surface opposite
affecting cells
properties like superhydrophilicity seems to open new perspectives in cell viability
attachment and proliferation is proposed in a paper [100], in which wax-impregnated cotton control.
fabrics were used as a microwell plate, easy to fabricate by a dipping and drying process.
Author Contributions: The manuscript was written through contributions of all authors. All authors have given
Microwell
approval platforms
to the final version are a widespread
of the standard
manuscript. M.F. in cell-based
and M.C.M., assays andwriting—review
conceptualization, drug screening&and, in
editing,
supervision, F.C.they
this case, writing—review
represent a&sustainable
editing. and environmentally friendly method. The influence of
surface
Funding: Thischemistry,
research did hydrophobicity, and roughness
not receive any specific grant fromwas investigated
funding agencies inonthecultured human skin
public, commercial, or
not-for-profit sectors.
fibroblasts. The study underlines a potential use for future cell-based assay platforms, usually
being made from non-biodegradable materials such as polystyrene or polyethylene or by the
soft lithography and photolithography technique.
j. In a recent work, a study on the influence of coating polyester fabric at different degrees of
hydrophobicity on a few mammalian cell viability lines was reported [101]. The composition
and structure of the mixed organic–inorganic coating with moderate to high water repellence
Colloids Interfaces 2019, 3, 48 12 of 16
Acknowledgments: This work was conducted under the umbrella of Cooperation Agreement between Faculty
of Pharmacy and Food Science (UB) and the Institute for Chemistry of Condensed Matter and Technologies for
Energy (ICMATE-CNR) (Codi GREC 18407, 2018-2021). M.C. Morán and M. Ferrari acknowledge the support of
UB-Mobility programme (OMPI-PR 1493, 2017-2018) for type A1 and type A2 grants, respectively. M. Ferrari
acknowledges the support of Compagnia San Paolo through the ANFISOL Project.
Conflicts of Interest: The authors declare no conflict of interest.
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