Chemically detachable polyelectrolyte multilayer platform for cell sheet engineering

Article abstract of DOI:10.1021/cm2024982

Human gingival fibroblasts (HGFs) cell sheets have a potential use for in vivo wound healing due to the ability of HGFs to adopt a contractile phenotype which is typically expressed during extracellular matrix tissue remodeling. For this purpose, we devel

Full text of DOI:10.1021/cm2024982

Article
pubs.acs.org/cm
Chemically Detachable Polyelectrolyte Multilayer Platform for Cell
Sheet Engineering
†
,‡
§,▽
†,‡
#
†,‡
Armelle Chassepot, Longcheng Gao,
Isabelle Nguyen, Alexandre Dochter, Florence Fioretti,
∥
⊥
§
#
†,‡,
Patrick Menu, Halima Kerdjoudj, Corinne Baehr, Pierre Schaaf, Jean-Claude Voegel, *
#
§
†,‡,
̈
Fouzia Boulmedais, Benoit Frisch, and Joelle Ogier *
†Institut National de la Sante
7085 Strasbourg Cedex, France
́
et de la Recherche Med
́
icale, INSERM Unite
́
977, Faculte
́
de Chirurgie Dentaire, 11 Rue Humann,
6
‡
Universite
§
́
de Strasbourg, Faculte
Laboratoire de Conception et Application de Molec
Pharmacie, 74 route du Rhin, 67401 Illkirch Cedex, France.
́
de chirurgie dentaire, 1 place de l’Ho
̂
pital 67000 Strasbourg
́
ules Bioactives, UMR 7199, CNRS/Universite
́
de Strasbourg, Faculte
́
de
∥
Group of Bioengineering (UMR CNRS 7561), UHP-Nancy 1, Faculte
Vandoeuvre-les-Nancy, France
Institut National de la Sante et de la Recherche Med
Marechal Juin, 51095 Reims Cedex, France
Institut Charles Sadron, Universite de Strasbourg, Centre National de la recherche scientifique, UPR 22, 23 rue du Loess, 67034
Strasbourg Cedex, France
de Medecine, 9 avenue de la foret de Haye 54500
́ ́ ̂
̀
⊥
́
́
icale, INSERM, UMR-S 926, IFR 53 URCA, UFR-Odontologie, 1 avenue du
́
#
́
*
S Supporting Information
ABSTRACT: Human gingival fibroblasts (HGFs) cell sheets have a potential use for
in vivo wound healing due to the ability of HGFs to adopt a contractile phenotype
which is typically expressed during extracellular matrix tissue remodeling. For this
purpose, we developed a chemically detachable platform based on poly(allylamine
hydrochloride)/poly(styrene sulfonate) multilayer film built on a sacrificial precursor
film which served as a substrate for HGF cell layer formation. The sacrificial precursor
film, based on disulfide-containing polycation and polyanion, is degradable under mild
conditions compatible for cell sheet detachment. Cellular viability and cell phenotype
analysis of HGF show that the designed platform promotes cell phenotype switch into
contractile phenotype, maintained after cell sheet lift-off. This contractile phenotype is
acquired by fibroblasts during in vivo wound healing and tissue remodeling. HGFs cell
sheet fragments, obtained by this detachment process, could be cultured later on
showing a good retention of the typical spindle-shape of differentiated cells after 10 days of culture. HGFs cell sheets have great
potential applications as autologous substrates for tissue repair and cellular synthetic platforms for research on connective tissue
diseases or evaluation of novel therapeutic agents.
KEYWORDS: layer-by-layer, disulfide bond, sacrificial platform, human gingival fibroblasts, autologous cell sheet, wound healing,
contractile phenotype
INTRODUCTION
impairment of cell functions. Strategies based on biodegradable
polymers (such as polyglycolic acid, poly-L-lactic acid,
polycaprolactone) provide promising results in vitro, but in
vivo data revealed severe limitations, together with too fast
degradation rates and promotion of acute inflammatory
■
Aimed at the maintenance and the recovery of native functions,
cell sheet engineering is emerging as a promising approach to
construct in vitro tissues or organs for transplantation into
damaged host tissues.
detachment were successfully developed such as the use of
thermo-responsive polymers,
matic digestion of the substrate, and magnetic force based or
electrochemical based detachment.
1
,2
Different strategies of cell sheet
14−16
responses.
3
−5
6,7
bioresorbable layers, enzy-
Polyelectrolyte multilayer (PEMs) films, obtained by the
alternate deposition of polycations and polyanions, emerged as
8
9
10−12
However, versatile and
mild methods for cell sheet development remain a challenge
due to persisting drawbacks such as irrelevant control over gene
expression induced by the detachment process. Enzymatic
treatment may induce damage to cell membranes by hydro-
lyzing various membrane-associated proteins, resulting in
Special Issue: Materials for Biological Applications
13
Received: August 22, 2011
Revised: December 14, 2011
Published: December 19, 2011
©
2011 American Chemical Society
930
dx.doi.org/10.1021/cm2024982 | Chem. Mater. 2012, 24, 930−937

Chemistry of Materials
Article
1
7,18
a simple approach to modify surfaces in a controlled way.
Poly(allylamine hydrochloride) and poly(styrene sulfonate)
(
PAH/PSS) films constitute a good substrate for differentiated
19−25
cell culture and then tissue engineering.
Those films even
accelerate the differentiation of circulating vascular progenitor
cells, harvested from peripheral blood into mature and
2
6,27
functional SMCs or ECs.
However, tissues formed from
cells cultured on PSS/PAH films are difficult to harvest while
preserving the cell−cell junction and their deposited extrac-
ellular matrix (ECM) compounds. Liao et al. used thermores-
ponsive PEMs for an efficient removal of human mesenchymal
2
8
Figure 1. Cytoskeleton visualization of HGFs by F-actin filament
stained with phalloidin (red labeling) observed by confocal
microscopy, after 14 days of culture on a (a) glass substrate and (b)
stem cells. Depending on the substrate on which PEM films
were deposited, free-standing films were lifted off using extreme
acidic conditions for silica or silicon substrates, acetone for
(
PAH/PSS) −PAH film.
10
29
cellulose substrates, and tetrahydrofuran or basic conditions
30
27
for polystyrene substrates. Organic solvent and extreme pH
are however incompatible with cell viability and cannot be used
to detach cellular sheets grown on PEM platforms. Buck et al.
reported the detachment of free-standing thin films based on
azalactone-containing polymer multilayers by immersion in a
PAH/PSS films. The expression of contractile proteins of
HGFs, seeded on glass and PAH/PSS films, has been checked
at both gene and protein level. The α-SMA gene, expressed by
cells, was investigated by quantitative real-time polymerase
chain reaction (qRT-PCR). These experiments included three
housekeeping gene controls as internal references, hypoxan-
thine phosphoribosyl transferase1 (HPRT1), beta-2 micro-
globulin (B2M), and glyceraldehyde phosphate dehydrogenase
31
mildly acidic aqueous environment.
In this study, we describe a novel biocompatible and easily
applicable chemical method for harvesting cell tissues in vitro.
PSS/PAH multilayer films, that served as a substrate for the
formation of cell layers, were built on a sacrificial precursor film.
This sacrificial film, based on disulfide-containing polycation
and polyanion, was degraded by a reducing agent under mild
conditions, compatible with cell viability and phenotype
stability. Cleavable disulfide bond under reductive conditions
is widely accepted and has been applied to produce porous
36,37
(
GAPDH). HPRT1, related to nucleotide metabolism, is
generally recognized to be independent of phenotype. B2M
and GAPDH were selected for the stability of their expression
40
during wound healing. Triplicate samples of cells, grown on
glass and (PAH/PSS) −PAH films, were analyzed after 5, 15,
10
and 30 days of culture. Figure 2 shows three to four times
3
2
33−35
multilayer films and in drug
or gene delivery.
Human gingival fibroblasts (HGFs) were used to produce
cell sheets because they are an easy source of autologous
primary cells with little donor-site morbidity. They are the main
population in oral mucosa of nonmyocyte cells dedicated to
establishment and maintenance of ECM and are useful graft
materials for the regeneration of alveolar bone and periodontal
3
8
ligaments. HGFs sheets have thus a potential use for in vivo
wound healing due to the HGFs ability to adopt a contractile
phenotype during tissue remodeling.
RESULTS AND DISCUSSION
■
Characterization of Human Gingival Fibroblasts Cell
Sheets Cultured on PAH/PSS Films. We first investigated
the HGFs behavior on PAH/PSS multilayers compared to a
glass substrate. Although the morphological observations of
HGFs performed after 7 days of culture did not show any
difference between (PAH/PSS) −PAH films and the glass
Figure 2. α-SMA mRNA quantification of HGFs cultivated on glass
substrate and (PAH/PSS) −PAH films during 5, 15, and 30 days.
10
1
0
substrate, the mitochondrial activity assessed by Alamar Blue
reagent showed a higher activity for cells cultivated on (PAH/
PSS) −PAH films (Figure S-1 in Supporting Information).
higher levels of α-SMA mRNA for cells cultivated on PEMs in
comparison to glass. PAH/PSS films dramatically promote the
expression of the α-SMA gene related to the contractile protein.
The expression of α-SMA contractile protein was checked by
immunocytochemistry staining. Almost all HGFs cultivated on
PAH/PSS films were positive to α-SMA marker after 15 days
10
Cytoskeleton staining showed that HGFs are well spread and
organized. Indeed, F-actin staining shows the presence of
polymerized actin fibers lying parallel to each other on both
glass and PEMs substrate (Figure 1). The density of F-actin
fibers seems higher on PAH/PSS films ended by PAH. All
these results demonstrate a better proliferation of HGFs on
PAH/PSS films compared to glass substrate.
(
(
Figure 3). Statistical analysis showed significant differences
p < 0.001 by Mann−Whitney test) in the number of HGFs
acquiring contractile phenotype on PAH/PSS films compared
to glass substrate from the fifth day of culture (Figure S-2 in
Supporting Information). After 15 days, the number of HGFs
with contractile phenotype are increased by about 36% when
cultured on PAH/PSS films compared to glass substrate
In vivo during wound healing and tissue remodeling, the
39
fibroblasts acquire a contractile phenotype by increasing the
expression of contractile proteins such as alpha smooth muscle
actin (α-SMA). Recently, Berthelemy et al. showed the high
expression of this protein for progenitor cells cultivated on
2
(control: 201 ± 25 per mm ; (PAH/PSS) −PAH film: 273 ±
10
9
31
dx.doi.org/10.1021/cm2024982 | Chem. Mater. 2012, 24, 930−937

Chemistry of Materials
Article
+
derived of poly(vinyl benzoic acid), PVBANH ) (Figure 5).
3
Both polymer backbones include disulfide bonds that can be
Figure 5. Chemical formulas of cleavable polyelectrolytes PVBACOO−
+
and PVBANH .
3
reduced by tris(2-carboxyethyl)phosphine (TCEP). They are
thus cleavable into small molecular fragments. Those
polyelectrolytes, soluble in water, were used to build a sacrificial
film on which a capping PAH/PSS film was deposited to detach
it later on by contact with TCEP.
Figure 3. Detection of α-SMA marker (green labeling) by
immunochemistry and nuclei blue counterstaining of HGFs cultured
(
a) on glass substrate and (b) (PAH/PSS) −PAH after 1, 5, and 15
10
−
+
PVBACOO /PVBANH3 multilayer films, built in physio-
logical conditions (150 mM NaCl, pH 7.4), grow linearly with
the number of deposited layers (Figure 6a). After contact with
3 mL of TCEP solution at 5 mM by three successive injections,
the film is dissolved at 96% in thickness. In the case of PEI−
days. The scale bars represent 100 μm.
2
3
3 per mm ). The contractile phenotype of HGFs is more
pronounced when seeded on PAH/PSS films compared to glass
substrate. In addition to the expression of the contractile
markers, tissue healing needs to have an appropriate ECM
−
+
(PVBACOO /PVBANH
)
12−(PSS/PAH) films, the contact
9
3
3
9
deposition. Then, the secretory activities of HGFs cultivated
on PAH/PSS films were investigated by immunocytochemistry.
At the beginning of wound healing, collagen I (COL1) is the
main component of the extracellular matrix produced by
fibroblasts. At the wound level, tissue homeostasis is controlled
by proteolytic enzymes such as matrix metalloproteinase-2
with TCEP leads to the degradation of 60% in film thickness
(Figure 6b). Atomic force microscopy (AFM) was used to
−
monitor the topography and the thickness of PVBACOO /
PVBANH3+ films with and without the capping PAH/PSS film.
−
+
3
As shown in Figure 7a, the PEI−(PVBACOO /PVBANH )
10
film is homogeneous and shows a roughness of 8 nm (Figure S-
3a in Supporting Information). After 30 min of contact with
TCEP, the film remained homogeneous (Figure S-3b) with a
roughness of 3 nm, but its dry thickness decreased from 32 to 5
(
MMP-2). HGFs cultivated on PAH/PSS express both pro-
COL1 and pro-MMP-2 proteins (Figure 4).
32
nm (Figure 7b). Chen et al. showed the formation of pores
on multilayered thin films containing a blend of reducible
polycations, based on cleavable disulfide bonds. In this work, a
nonreducible polyanion was employed. No pore formation was
observed in our system which is composed of both reducible
polycations and polyanions. TCEP treatment leads to almost
complete film dissolution. When a capped PAH/PSS film is
−
+
built on top of PVBACOO /PVBANH , a homogeneous film
3
is obtained with a higher roughness, about 14 nm compared to
the 3 nm of the noncapped film (Figure S-3c in Supporting
Figure 4. Detection of (a) pro-COL1 (green labeling) and (b) pro-
MMP2 (green labeling) by immunochemistry and blue counter-
staining of HGFs nuclei after 15 days culture on (PAH/PSS) −PAH
−
Information). TCEP treatment of the PEI−(PVBACOO /
+
PVBANH ) −(PSS/PAH) film induced a homogeneous
10
3
10
10
film.
decrease of the dry thickness from 58 to 30 nm with almost no
change in roughness (Figure 7c,d, Figure S-3d in Supporting
Information). Finally, the (PVBANH /PVBACOO ) −
(PAH/PSS−Rhodamine) −PAH architecture was built and
+
−
These results confirm an advantage for PAH/PSS films in
terms of fibroblast functionality (contractility and homeostasis
properties). These properties are essential for connective tissue
3
10
10
incubated for 10 min in 5 mM TCEP solution. Fluorescent
microscopy images confirmed the delamination of the PEM
and the presence of PSS−Rhodamine on the released
membrane after TCEP treatment (Figure S-4 in Supporting
Information).
41
remodeling during normal wound healing. Production of cell
sheets expressing an accurate phenotype would be of interest as
substrate for tissue repair.
Buildup and Degradation of Disulfide Containing
Polyelectrolyte Films. PAH/PSS films ended by PAH have
been shown to be appropriate films for HGFs culture and even
suitable platforms for cells sheet engineering. In order to detach
the HGFs cell layer obtained on PAH/PSS films, we
synthesized by polymerization cleavable polyanion (poly(vinyl
Detachment of Cell Sheet and Cell Phenotype
Characterization. HGFs viability remains similar for PAH/
PSS films built either directly on the glass substrate or on a
+
3
−
PVBANH /PVBACOO sacrificial platform (Figure S-1 in
Supporting Information). To test the cytotoxicity of the
reductive agent, HGFs previously cultured on glass substrate
−
benzoic acid, PVBACOO ) and polycation (polycationic
9
32
dx.doi.org/10.1021/cm2024982 | Chem. Mater. 2012, 24, 930−937

Chemistry of Materials
Article
−
+
−
+
Figure 6. Thickness evolution of (a) PEI−(PVBACOO /PVBANH ) and (b) PEI−(PVBACOO /PVBANH ) −(PSS/PAH) multilayer films,
3
7
3
12
9
determined by QCM, as a function of the last layer deposited, followed by four injections of 5 mM TCEP solution. A represents the polyanion
−
+
PVBACOO and B represents the polycation PVBANH .
3
for 7 days were incubated with a 5 mM TCEP solution (Figure
S-5 in Supporting Information). TCEP solution does not show
any cytotoxicity to HGFs up to 30 min of contact.
could be necessary to engineer specific demanding cell
phenotypes. Moreover, the use of a chemically degradable
platform does not need a dedicated electrochemical or
magnetic force apparatus.
+
−
After 30 days of culture on (PVBANH /PVBACOO ) −
3
10
(
PAH/PSS) −PAH, HGFs layers were incubated with 5 mM
10
TCEP for less than 30 min. At the beginning of the process, the
detached part of the HGFs cell sheets (II) remained connected
to the cell layer (III) that still adhered to the surface (Figure
CONCLUSION
■
In this work, we described a platform to produce detachable
human autologous cell sheets. The obtained results demon-
strate the ability of the platform not only to drive HGFs
adhesion and proliferation but also to change their morphology
to spread and express activation-dependent phenotypes.
8
a). Figure 8b shows a macroscopic image of the obtained free-
standing cell sheet (II′) after total detachment. HGFs sheets
were then characterized by immunochemical detection of
COL1 and α-SMA. Immunostaining revealed the preservation
of secreted pro-COL1 and α-SMA expression, suggesting that
TCEP treatment had no effect on HGF tissue integrity (Figure
+
−
Specifically, (PVBANH /PVBACOO ) −(PAH/PSS) −
3
10
10
PAH platform promotes HGFs conversion to the contractile
phenotype, which is maintained after cell sheet lift-off. In this
respect, the method could provide fibroblast-seeded substrates
having the potential to reorganize ECM and thereby to confer
adaptation to tissue injury. As such, the construct described
here could be envisioned as substrate for tissue repair. It could
also serve a synthetic cell platform for research on connective
tissue disease and evaluation of novel therapeutic agents.
9
).
+
−
A (PVBANH /PVBACOO ) −(PAH/PSS−Rhod-
3
10
amine) −PAH film was also used as a substrate for HGFs
10
culture for 30 days and underwent the TCEP treatment. Figure
S-6 in Supporting Information shows the presence of PSS−
Rhodamine in the detached cell sheet showing the integrity of
PAH/PSS film. PAH/PSS films constitute a good substrate for
1
9−25
differentiated cell culture
but also for the differentiation of
EXPERIMENTAL SECTION
2
6,27
■
stem cells into mature and functional SMCs or ECs.
Synthesis of Polymers. Thin layer chromatography (TLC) was
performed on precoated plates (0.25 mm Silica Gel 60, F254, Merck,
Darmstadt, Germany). Products were purified by chromatography
over silica gel (Silica Gel 60, 40−63 μm, Merck, Darmstadt, Germany).
NMR spectra were recorded on Bruker 300 MHz Avance DPX and
400 MHz Avance 3 ultrashield+ instruments. 1H and C NMR
chemical shifts δ are reported in ppm relative to their standard
42
Recently, Guillaume-Gentil et al. report the use of PAH/PSS
thin films as substrate for the growth and subsequent release
under electrochemical control of mesenchymal stem cell sheets
that keep their mesodermal plasticity. These studies and our
results show a good biocompatibility of PAH/PSS films.
Cell migration is an important characteristic for tissue
healing. According to the explants technique, we have seeded
fragments of the detached cell sheet on a new glass substrate.
After 30 days of HGFs culture (Figure 10a) and detachment,
fragments of the detached cell sheet were put in culture to
check the compatibility of the detachment procedure (Figure
13
1
reference ( H: CHCl at 7.27 ppm, DMF (−CH ) at 2.88 ppm, tert-
3
3
13
butanol at 1.24 ppm; C: CDCl at 77.0 ppm). Mass Spectra (MS)
3
were recorded on an Agilent MSD SQ 1200SL instrument, using
multimode APCI/Electrospray (ESI) mode and coupled to HPLC
1
200SL. Mass data are reported in mass units (m/z). Unless otherwise
specified, all chemicals were reagent grade and were used as received:
sulfur flowers and 4-vinylbenzoic acid were purchased from Alfa Aesar
(Bischeim, France) sodium sulfide, N-hydroxysuccinimide, N-Boc-2,2′-
1
0b). As early as 3 days of culture, cells migrated from the cell
sheet fragment (I) to the glass substrate. After 10 days of
culture, outgrowth of cells from the cell sheet fragments was
observed with retention of the typical spindle-shape of
differentiated cells (Figure 10c,d). These results demonstrate
that the detached cells kept their migration property.
A major advantage of this chemically detachable platform,
based on PEMs, concerns the ability to be equipped by an
additional multiple functionalization with growth factors that
(ethylenedioxy)diethylamine were all purchased from Aldrich (St.
Quentin Fallavier, France), and N,N′-dicyclohexyl-carbodiimide was
purchased from Acros (Thermo Fisher Scientific, Strasbourg, France).
Polymerization steps of disulfide-containing polyelectrolytes are
depicted in Scheme 1.
Synthesis of BVBA. A solution of bromine (360 μL, 6.95 mmol)
in chloroform (5 mL) was added dropwise at 0 °C to a solution of
vinylbenzoic acid (VBA) (1 g, 6.74 mmol) in chloroform (20 mL).
9
33
dx.doi.org/10.1021/cm2024982 | Chem. Mater. 2012, 24, 930−937

Chemistry of Materials
Article
−
+
Figure 7. Typical AFM 3D images, obtained in contact mode in dry state, of PEI−(PVBACOO /PVBANH ) film (a) before and (b) after TCEP
3
10
−
+
treatment (5 mM, 30 min) and of PEI−(PVBACOO /PVBANH ) −(PSS/PAH) film (c) before and (d) after TCEP treatment (5 mM, 30 min).
3
10
10
The films were scratched before observation.
The solution was strirred at room temperature for 4 h. Chloroform
was removed under vacuum to give 2.05 g (98%) of BVBA as a white
g, 3.57 mmol) and sodium hydroxide (143 mg, 3.57 mmol) in water
(10 mL) was added. The mixture was stirred at room temperature for
1
solid. H NMR (300 MHz, CDCl ) δ 4.05 (m, 2H, CH ), 5.16 (dd, J =
3
2
24 h. The polymer was precipitated in ethanol to give 500 mg as a
5
8
4
1
.2, 11.0 Hz, 1H, CHBr), 7.52 (d, J = 8.4 Hz, 2H, CH ), 8.13 (d, J =
1
Ar
white solid. H NMR (300 MHz, D O, reference tert-butanol) δ 2.5−
13
2
.4 Hz, 2H, CH ); C NMR (100 MHz, CDCl ) δ 34.43 (CH ),
Ar
3
2
4
.5 (m, 3nH, CHCH ), 7.27 (m, 2nH, CH ), 7.87 (m, 2nH, CH ). n
2 Ar Ar
9.30 (CHBr), 128.14 (CH ), 130.06 (C CHBr), 130.98 (CH ),
Ar
Ar
Ar
−
is the degree of polymerization (DPn). PVBACOO has a mean
44.56 (C COOH).
Ar
3
−
molecular weight of M = 28 × 10 g/mol and a polydispersity M /M
Synthesis of Polymer PVBACOO . A solution of sulfur flowers
114 mg, 3.57 mmol) and sodium sulfide (278 mg, 3.57 mmol) in
water (10 mL) was stirred 2 h at 70 °C. Then a solution of BVBA (1.1
w
w
n
(
= 1.69, determined by Steric Exclusion Chromatograpy - Multi Angle
Light Scattering (SEC-MALS).
9
34
dx.doi.org/10.1021/cm2024982 | Chem. Mater. 2012, 24, 930−937

Chemistry of Materials
Article
Synthesis of BVBANHBoc. To a solution of BVBA (872 mg, 2.83
mmol) in dichloromethane (40 mL) were added N,N′-dicyclohex-
ylcarbodiimide (636 mg, 3.084 mmol) and N-hydroxysuccinimide
(
354 mg, 3.084 mmol), and the solution was stirred at room
temperature for 30 min. A solution of N-Boc-2,2′-(ethylenedioxy)-
diethylamine (656 mg, 2.83 mmol) in dichloromethane (10 mL) was
added dropwise and kept under stirring at room temperature for 15 h.
After removing the precipitate, the residue was purified by silica-gel
chromatography to afford 544 mg (40%) of BVBANHBoc as a
1
colorless oil. H NMR (300 MHz, CDCl ) δ 1.44 (s, 9H, C(CH ) ),
3
3 3
3
.29 (m, 2H, CH NHBoc), 3.53−3.72 (m, 10H, CH PEG), 3.97−
Figure 8. Images obtained by optical (a) microscopy and (b)
2
2
4
.09 (m, 2H, CH Br), 4.98 (NH), 5.14 (dd, J = 5.2, 10.7 Hz, 1H,
macroscopy of a HGFs cell sheet, cultured for 30 days on a
2
1
3
+
−
CHBr), 6.77 (NH), 7.47 (m, 2H, CH ), 7.81 (m, 2H, CH );
C
(
PVBANH /PVBACOO ) −(PAH/PSS) −PAH film, recovered
Ar
Ar
3
10
10
NMR (100 MHz, CDCl ) δ 28.9 ((CH ) ), 34.9 (CH Br), 39.8−40.3
from mild chemical treatment during 30 min: the areas represent
respectively (I) the glass substrate, (II and II′) free-standing HGFs cell
sheet, and (III) HGFs layer.
3
3
3
2
(
1
NHCH2 and CH NH), 49.8 (CHBr), 70.6 (CH PEG), 127.8−
2
2
28.1(CH ), 135.1 (C CHBr), 141.9 (C COOH), 156.1 and 167.0
Ar Ar Ar
+
(
CO); LR-MS (ESI) calcd for C H Br N NaO [(M+Na) ]:
20 30 2 2 5
5
61.0399; found 561.0419.
Synthesis of Polymer PVBANHBoc. A solution of sulfur flowers
(
(
32 mg, 1.00 mmol) and sodium sulfide (78 mg, 1.00 mmol) in water
6 mL) was stirred 2 h at 70 °C. The reaction mixture was added
dropwise to a solution of BVBANHBoc (540 mg, 1.034 mmol) in
DMF (10 mL). The solution was strirred at room temperature for 24
h. The solution was diluted in ethanol (20 mL), and the polymer was
precipited into chilled diethyl ether (500 mL) to give 264 mg (64%) of
1
the polymer PVBANHBoc as white a solid. H NMR (300 MHz, D O,
2
reference DMF) δ 1.30−1.38 (m, 9nH, C(CH ) ), 3−3.81 (m, 15nH,
3
3
CH PEG, CHCH S), 7.19−7.92 (m, 4nH, CH ). n is the degree of
2
2
Ar
polymerization (DPn).
Synthesis of polymer PVBANH . To a solution of polymer
+
3
PVBANHBoc (260 mg, 0.64 mmol) in dichloromethane (20 mL) was
added trifluoroacetic acid (10 mL), and the solution was stirred at
room temperature for 24 h. The solvent was removed by vacuum, and
water (20 mL) was added to dissolve the polymer. After filtration and
+
lyophilization, the polymer PVBANH3 was found as a slightly green
1
solid, 290 mg (100%). H NMR (300 MHz, D O, reference tert-
2
butanol) δ 3.13 (s, 2nH, −CH NH ), 3.57 (m, 10nH, CH PEG), 2.7−
2
2
2
4
.5 (bm, 3nH, CHCH S), 7.25 (bm, 2nH, CH ), 7.75 (bm, 2nH,
Ar
2
A
r
Figure 9. Detection of pro-COL1 (red labeling) and α-SMA (green
CH ). n is the degree of polymerization (DPn).
labeling) markers by immunochemistry and blue counterstaining of
Chemicals and Polyelectrolyte Solutions. Poly(allylamine
+
−
HGFs nuclei after 30 days culture on a (PVBANH /PVBACOO ) −
4
3
10
hydrochloride) (PAH, Mw = 7 × 10 g/mol), poly(sodium 4-
(
PAH/PSS) −PAH film after detachment (a, b, and d) and (c) before
4
10
styrenesulfonate) (PSS, M = 7.0 × 10 g/mol), poly(ethylene imine)
w
detachment of the cell sheet.
4
(PEI, Mw = 75 × 10 g/mol), (4-(2-hydroxyethyl)-1-piperazine
ethanesulfonic acid (Hepes), and Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) were all purchased from Sigma (St. Quentin
Fallavier, France). 10 mM Hepes−150 mM NaCl buffer at pH 7.4 was
prepared to dissolve polyelectrolytes and TCEP using ultrapure water
(
Milli-Q- plus system, Millipore) with a resistivity of 18.2 MΩ·cm. All
polyelectrolyte solutions were prepared at 0.5 mg/mL by dissolution
of adequate amounts of polyelectrolyte powders in Hepes−NaCl
buffer. The solutions were freshly prepared before use.
Quartz Crystal Microbalance. The construction of multilayer
films was monitored in situ by quartz crystal microbalance with
dissipation monitoring QCM (Q-Sense E4, Gotenborg, Sweden). The
̈
QCM technique consists of measuring the resonance frequency
changes (Δf ) induced by polyelectrolyte adsorption on a quartz
ν
crystal. The quartz crystal is excited at its fundamental frequency (5
MHz), and the measurements are performed at the first, third, fifth,
and seventh overtones (denoted by ν = 1, 3, 5, and 7, respectively)
corresponding to about 5, 15, 25, and 35 MHz, respectively. Changes
in the resonance frequencies, Δf , once the excitation is switched off
ν
are measured at these four frequencies. The quartz crystals used here
are coated with a ∼50 nm SiO film. The sensor is fixed in removable
2
flow modules with inlet and outlet and maintained at 22 °C. In each
experiment, 600 μL of Hepes−NaCl solution were injected into the
measurement cell. After stabilization of the signals (shift in reduced
frequency typically lower than 0.5 Hz/min), 600 μL of the polycation
solution dissolved in a Hepes−NaCl buffer was injected. This solution
was left in the cell for 5 min, rinsed with the Hepes−NaCl buffer, and
left again for 5 min. During the whole process, the frequency shifts
Figure 10. Phase contrast microscopy images of HGFs cell sheet after
+
−
1
5 days of culture on a (PVBANH /PVBACOO ) −(PAH/PSS) −
3
10
10
PAH film (a) before detachment, (b) detached and transferred on a
new substrate, (c) after 3 days of culture, and (d) after 10 days of
culture. (I) represents the initially detached cell sheet.
9
35
dx.doi.org/10.1021/cm2024982 | Chem. Mater. 2012, 24, 930−937

Chemistry of Materials
Scheme 1. Schematic Representation of Synthesis of Polymer PVBACOO and PVBANH
Article
−
+
3
were continuously recorded as a function of time. The same procedure
was used for the deposition of the polyanion. The construction was
pursued by alternate depositions of polycation and polyanion. A
positive shift in the opposite direction of the normalized frequency
Immunocytochemical Detection of Collagen Type I, F-Actin,
MMP2 and Alpha-Actin. Cells were fixed with 3.7% paraformalde-
hyde for 10 min at 4 °C, permeabilized in 0.25% Triton X-100 in PBS
for 10 min, and blocked in 1% BSA−PBS for 30 min. Thereafter, cells
were incubated for 3 h at room temperature with rabbit anticollagen
type I (COL1) antibody (Ab-cam), mouse monoclonal antihuman
smooth muscle α-actin (α-SMA) (Dako), or antimetalloprotease 2
shift, −Δf /ν, can be associated, in first approximation, with an
ν
increase of the mass adsorbed on the crystal. In our case, the
normalized frequency shifts (15, 25, and 35 MHz) are superimposed,
and then the Sauerbrey’s relation can be applied to determine the
(
MMP2) at the dilutions of 1/200 and 1/50, respectively. Slides were
then extensively washed with PBS, and a secondary antibody
Invitrogen Alexa fluor antirabbit IgG or Rockland goat polyclonal
43
thickness adsorbed.
(
Buildup of Multilayered Films on Glass Coverslips.
Polyelectrolyte multilayer films have been prepared in 24 wells plates
on glass coverslips (VWR, Strasbourg, France) pretreated with 0.1 M
SDS for 15 min followed by 0.1 M HCl for 15 min at 100 °C and
rinsed with extensively distilled water between each treatment. 300 μL
of polycation solution were deposited for 10 min on glass coverslips
followed by three rinses with Hepes−NaCl buffer. Then, 300 μL of
polyanionic solution were added for 10 min followed by three rinses.
These steps were repeated according to the chosen film scheme,
FITC antimouse IgG, respectively) was added at a dilution of 1/1000
and incubated for 2 h at room temperature in the dark, followed by
nuclear counterstaining with DAPI (50 ng/mL) (Invitrogen)
incubated for 2 min at room temperature. F-actin filaments were
observed by incubation of cells for 30 min at room temperature with 5
−
5
×
(
10 mg/mL phalloidin-tetramethylrhodamine B isothiocyanate
Sigma-Aldrich). Washed slides were mounted on blades with DAKO
fluorescent mounting medium (DAKO), and fluorescence distribution
was examined by means of an inverse fluorescence microscope
+
−
typically (PAH/PSS) −PAH and (PVBANH /PVBACOO ) −
n
3
m
(
Axiovert, Zeiss).
(
PAH/PSS) −PAH films.
n
Quantitative Real-Time Reverse Transcriptase Polymerase
Cell Culture on Multilayer Films. Gingival fibroblasts (HGFs)
Chain Reaction (qRT-PCR). Total RNA was isolated from the cells
on each well after trypsin extraction with the RNeasy Mini Kit from
QIAGEN. The amount of RNA in each sample was quantified using a
NanoDrop spectrophotometer (Thermo Scientific) and adjusted at
were isolated from human gingival connective tissue of healthy
individuals according to a protocol approved by the ethics committee
for patient protection of CPP Strasbourg Hospitals. Gingival tissues
were obtained from gingival samples without inflammation removed
for tooth extraction purposes, cut into small pieces, placed into culture
dishes, and incubated in a humidified atmosphere of 95% air and 5%
1
50 ng/μL. cDNA strand was generated from 10 μL of total RNA
using the Superscript II kit from Invitrogen. The amplification of
cDNAs was performed in separate reactions of 40 cycles each in a
Biorad PCR system (Bio-Rad MyiQ 2 Two-Color Real-Time PCR
TM
CO at 37 °C for 30 min. Then, culture medium (DMEM with 20%
2
FBS, 100 μg/mL penicillin-streptomycin, and 2 μg/mL Fungizone,
Gibco) was added. After reaching confluency, the cells were harvested
using 0.25% trypsin. HGFs were used between the fourth and sixth
passage. For phenotype characterization, HGFs were seeded in 24
wells plates at the density of 3 × 10 cells/cm over glass coverslips
covered with different architectures.
Detection System) using the Quantitec SYBR Green PCR Kit and
gene-specific primer pairs (QuantiTec Primer) from QIAGEN for α-
SMA and the beta2 microglobulin (BM2), hypoxanthine phosphor-
ibosyl transferase1 (HPRT1), and glyceraldehyde phosphate dehydro-
genase (GAPDH) housekeeping gene controls. Gene expression level
stability was analyzed by the GeNorm algorithm. The experiment was
repeated three times, and the analysis of marker expression was
4
2
Detachment of HGFs Cell Sheet. After 30 days of HGFs culture
+
−
TM
on (PVBANH /PVBACOO ) −(PAH/PSS) −PAH film, the cell
performed using the iQ 5 Optical System Software.
3
10
10
layer was detached by contact with TCEP for less than 30 min.
Tweezers were used to peel the cell sheet. After detachment, the cell
sheet was immersed in the cell culture medium.
ASSOCIATED CONTENT
Supporting Information
■
*
S
Viability Assay. The measurement of cell viability was done by the
AlamarBlue assay (Biosource International). This assay is based on the
reduction of the blue, nonfluorescent dye resazurin to the pink and
fluorescent dye resorufin by living cells. Viability was assessed for
different times. After rinsing cells with 10% PBS reagent in complete
medium, they were incubated for 3 h. After incubation, the optical
densities (OD) at 570 and 630 nm were determined with a microplate
reader. The percentage of reduction of AlamarBlue was calculated
according to the procedure provided by the manufacturer.
Viability of HGFs on different multilayer films, Number of
HGFs with α-SMA markers cultivated at various days,
Topography of different multilayer films observed by AFM,
free standing PAH/PSS membrane observed by fluorescence
microscopy, viability of HGFs in the presence of TCEP and
HGFs cell sheet with fluorescently labeled PSS observed by
fluorescence microscopy. This material is available free of
charge via the Internet at http://pubs.acs.org.
9
36
dx.doi.org/10.1021/cm2024982 | Chem. Mater. 2012, 24, 930−937

Chemistry of Materials
Article
(
25) Thebaud, N. B.; Bareille, R.; Daculsi, R.; Bourget, C.; Remy, M.;
AUTHOR INFORMATION
Corresponding Author
E-mail: jean-claude.voegel@medecine.u-strasbg.fr, joelle.
ogier@medecine.u-strasbg.fr.
■
Kerdjoudj, H.; Menu, P.; Bordenave, L. Acta Biomater. 2010, 6 (4),
437−1445.
26) Berthelemy, N.; Kerdjoudj, H.; Gaucher, C.; Schaaf, P.; Stoltz, J.
1
(
*
F.; Lacolley, P.; Voegel, J.-C.; Menu, P. Adv. Mater. 2008, 20, 2674−
2679.
(
Lacolley, P.; Stoltz, J. F.; Voegel, J. C.; Menu, P. PLoS One 2009, 4,
e5514.
(
Present Address
Key Laboratory of Bioinspired Smart Interfacial Science and
Technology of Ministry of Education, School of Chemistry and
Environment, Beihang University, Beijing, 100191, P.R. China.
▽
27) Berthelemy, N.; Kerdjoudj, H.; Schaaf, P.; Prin-Mathieu, C.;
28) Liao, T. Q.; Moussallem, M. D.; Kim, J.; Schlenoff, J. B.; Ma, T.
Biothechnol. Prog. 2010, 26 (6), 1705−1713.
(29) Mamedov, A. A.; Kotov, N. A. Langmuir 2000, 16 (13), 5530−
5533.
ACKNOWLEDGMENTS
The authors acknowledge financial support from ANR E-
DETACHPEM BLAN08-1_315174, ANR SUBVACEL ANR-
7-TECSAN-022-01, and CNRS Prise de risque 2008 (Projet
Vaisseau In situ) grants.
■
(30) Lavalle, P.; Boulmedais, F.; Ball, V.; Mutterer, J.; Schaaf, P.;
0
Voegel, J.-C. J. Membr. Sci. 2005, 253 (1−2), 49−56.
(31) Buck, M. E.; Lynn, D. M. Langmuir 2010, 26 (20), 16134−
1
(
2
(
6140.
32) Chen, J.; Xia, X. M.; Huang, S. W.; Zhuo, R. X. Adv. Mater.
007, 19 (7), 979−983.
33) Saito, G.; Swanson, J. A.; Lee, K. D. Adv. Drug. Delivery Rev.
REFERENCES
■
(
1) Shimizu, T.; Yamato, M.; Kikuchi, A.; Okano, T. Biomaterials
003, 24 (13), 2309−2316.
2
2
(
003, 55 (2), 199−215.
(
1
(
2) Nichol, J. W.; Khademhosseini, A. Soft Matter 2009, 5 (7), 1312−
319.
3) Yang, J.; Yamato, M.; Kohno, C.; Nishimoto, A.; Sekine, H.;
Fukai, F.; Okano, T. Biomaterials 2005, 26 (33), 6415−6422.
4) Okano, T.; Yamada, N.; Okuhara, M.; Sakai, H.; Sakurai, Y.
Biomaterials 1995, 16 (4), 297−303.
5) Elloumi-Hannachi, I.; Yamato, M.; Okano, T. J. Int. Med. 2010,
67 (1), 54−70.
6) Iwasaki, K.; Kojima, K.; Kodama, S.; Paz, A. C.; Chambers, M.;
Umezu, M.; Vacanti, C. A. Circulation 2008, 118 (14), S52−S57.
7) Chong, M. S. K.; Chan, J.; Choolani, M.; Lee, C. N.; Teoh, S. H.
Biomaterials 2009, 30 (12), 2241−2251.
8) Nagai, N.; Yunoki, S.; Satoh, Y.; Tajima, K.; Munekata, M. J.
Biosci. Bioeng. 2004, 98 (6), 493−496.
9) Ito, A.; Ino, K.; Kobayashi, T.; Honda, H. Biomaterials 2005, 26
31), 6185−6193.
10) Guillaume-Gentil, O.; Akiyama, Y.; Schuler, M.; Tang, C.;
Textor, M.; Yamato, M.; Okano, T.; Voros, J. Adv. Mater. 2008, 20 (3),
60−565.
11) Inaba, R.; Khademhosseini, A.; Suzuki, H.; Fukuda, J.
Biomaterials 2009, 30 (21), 3573−3579.
12) Seto, Y.; Inaba, R.; Okuyama, T.; Sassa, F.; Suzuki, H.; Fukuda,
J. Biomaterials 2010, 31 (8), 2209−2215.
13) Isenberg, B. C.; Tsuda, Y.; Williams, C.; Shimizu, T.; Yamato,
M.; Okano, T.; Wong, J. Y. Biomaterials 2008, 29 (17), 2565−2572.
14) Bostman, O.; Pihlajamaki, H. Biomaterials 2000, 21 (24), 2615−
621.
15) Gilbert, T. W.; Stewart-Akers, A. M.; Badylak, S. F. Biomaterials
007, 28 (2), 147−150.
16) Falco, E. E.; Patel, M.; Fisher, J. P. Pharm. Res. 2008, 25 (10),
34) Zelikin, A. N.; Quinn, J. F.; Caruso, F. Biomacromolecules 2006,
7
(
(1), 27−30.
35) El-Sayed, M. E. H.; Hoffman, A. S.; Stayton, P. S. J. Controlled
Release 2005, 101 (1−3), 47−58.
36) McKenzie, D. L.; Smiley, E.; Kwok, K. Y.; Rice, K. G.
Bioconjugate Chem. 2000, 11 (6), 901−909.
37) Park, Y.; Kwok, K. Y.; Boukarim, C.; Rice, K. G. Bioconjugate
Chem. 2002, 13 (2), 232−239.
38) Nakajima, K.; Abe, T.; Tanaka, M.; Hara, Y. J. Periodontal Res.
008, 43 (6), 681−688.
39) Hinz, B.; Celetta, G.; Tomasek, J. J.; Gabbiani, G.; Chaponnier,
C. Mol. Biol. Cell 2001, 12, 2730−2741.
40) Turabelidze, A.; Guo, S. J.; DiPietro, L. A. Wound. Rep. Reg.
010, 18 (5), 460−466.
41) Tomasek, J. J.; Gabbiani, G.; Hinz, B.; Chaponnier, C.; Brown,
R. A. Nat. Rev. Mol. Cell Biol. 2002, 3 (5), 349−363.
42) Guillaume-Gentil, O.; Semenov, O. V.; Zisch, A. H.;
Zimmermann, R.; Voros, J.; Ehrbar, M. Biomaterials 2011, 32 (19),
376−4384.
43) Sauerbrey, G. Z. Phys. 1959, 155, 206−222.
(
(
(
2
(
(
(
2
(
(
(
(
2
(
(
(
(
(
5
(
4
(
(
(
(
2
(
2
(
2
(
(
348−2356.
17) Decher, G. Science 1997, 277 (5330), 1232−1237.
18) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir
1
997, 13 (13), 3422−3426.
19) Tryoen-Toth, P.; Vautier, D.; Haikel, Y.; Voegel, J. C.; Schaaf,
P.; Chluba, J.; Ogier, J. J. Biomed. Mater. Res. 2002, 60 (4), 657−67.
20) Boura, C.; Menu, P.; Payan, E.; Picart, C.; Voegel, J.-C.; Muller,
S.; Stoltz, J. F. Biomaterials 2003, 24, 3521−3530.
21) Boura, C.; Muller, S.; Vautier, D.; Dumas, D.; Schaaf, P.; Voegel,
J. C.; Stoltz, J. F.; Menu, P. Biomaterials 2005, 26 (22), 4568−4575.
22) Moby, V.; Boura, C.; Kerdjoudj, H.; Voegel, J. C.; Marchal, L.;
Dumas, D.; Schaaf, P.; Stoltz, J. F.; Menu, P. Biomacromolecules 2007, 8
7), 2156−2160.
23) Kerdjoudj, H.; Boura, C.; Moby, V.; Montagne, K.; Schaaf, P.;
Voegel, J. C.; Stoltz, J. F.; Menu, P. Adv. Funct. Mater. 2007, 17 (15),
667−2673.
24) Kerdjoudj, H.; Berthelemy, N.; Rinckenbach, S.; Kearney-
(
(
(
(
(
(
2
(
Schwartz, A.; Montagne, K.; Schaaf, P.; Lacolley, P.; Stoltz, J. F.;
Voegel, J. C.; Menu, P. J. Am. Coll. Cardiol. 2008, 52 (19), 1589−1597.
9
37
dx.doi.org/10.1021/cm2024982 | Chem. Mater. 2012, 24, 930−937