Bone Formation Mediated by Synergy-Acting Growth Factors
Embedded in a Polyelectrolyte Multilayer Film**
By Andree Dierich, Erell Le Guen, Nadia Messaddeq, Jean-François Stoltz, Patrick Netter,
Pierre Schaaf, Jean-Claude Voegel, and Nadia Benkirane-Jessel*
For decades, the treatment of degenerative cartilage and
bone diseases has been a challenge for orthopedic surgeons
because of the apparent inability of cartilage and bone to repair itself. There is no effective therapy available and patients
can only be helped by surgical joint replacement. An inherent
major concern is the limited availability of autografts, which
significantly reduces the choice of treatable defects. However,
new approaches to cell grafting are being developed in this
field: increased yields of cells are achieved through the use of
bioreactors and growth-factor administration, such as transforming growth factor b1 (TGFb1) and bone morphogenetic
proteins (BMPs).[1,2] Additionally, stem cells are being discovered as a new source of transplantable material.
Embryonic stem (ES) cells represent a valuable source for
cell transplantation because their characteristic features include an unlimited self-renewing capacity and a multilineage
differentiation potential.[3,4] In fact, ES-derived glial precur-
–
[*] Dr. N. Benkirane-Jessel, E. Le Guen, Dr. J.-C. Voegel
Faculté de Chirurgie Dentaire de L’Université Louis Pasteur
Institut National de la Santé et de la Recherche Médicale, Unité
595, Faculté de Médecine
11 rue Humann, 67085 Strasbourg Cedex (France)
E-mail: [email protected]
Dr. A. Dierich, Dr. N. Messaddeq
Institut de Génétique et de Biologie Moléculaire et
Cellulaire (IGBMC)
Institut Clinique de la Souris (ICS), CNRS/INSERM/ULP
Collège de France
BP 10142, Strasbourg (France)
Prof. J.-F. Stoltz, Prof. P. Netter
Unite Mixte de Recherches 7561
Centre National de la Recherche Scientifique
Université Henri Poincaré Nancy 1, Faculté de Médecine
Vandoeuvre les Nancy
54000 Nancy (France)
Prof. P. Schaaf
Institut Charles Sadron (CNRS/ULP)
6 rue Boussingault, 67083 Strasbourg Cedex (France)
[**] We thank Professor Pierre Chambon (IGBMC, ICS, Collège de
France, Strasbourg) for helpful discussions. This work was supported by the “Fondation de L’Avenir pour la Recherche Médicale Appliquée” and the “Ligue contre le Cancer (Région Alsace)”. N.J. is
indebted to Bernard Senger for many fruitful and stimulating discussions and to Raphael Darcy (UCD, Dublin) for the generous gift
of Cyclodextrin. Supporting Information is available online from
Wiley InterScience or from the author.
Adv. Mater. 2007, 19, 693–697
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DOI: 10.1002/adma.200601271
sors and cardiomyocytes have been successfully transplanted,
integrated, and shown to be functionally active in the transplantation site.[5,6] The yield of differentiation of ES cells into
an intended lineage can be greatly enhanced by the addition
of growth factors or induction substances. Although protocols
for the differentiation of cardiomyocytes, neuronal cell types,
insulin-producing cells, or adipocytes from ES cells have been
available for many years,[7–10] only recently has their differentiation into elements of the skeleton been reported.[11–14]
In recent years, considerable effort has been devoted to the
design and controlled fabrication of structured materials with
functional properties.[15] The layer-by-layer buildup of polyelectrolyte multilayer (PEM) films from oppositely charged
polyelectrolytes[16] offers new opportunities for the preparation of functionalized biomaterial coatings. This technique
allows the preparation of supramolecular nanoarchitectures
exhibiting specific properties in terms of control of cell activation[17–20] and may also play a role in the development of local
drug-delivery systems.[21–23] Peptides and proteins chemically
bound to polyelectrolytes, or adsorbed or embedded in PEM
films, have been shown to retain their biological activity.[23]
Cyclodextrins (CDs) constitute a group of cyclic oligosaccharides that have been shown to improve the bioavailability
of many drugs by forming inclusion complexes with them.[22]
CDs and certain of their derivatives also play an important
role in drug formulation because of their effect on solubility,
dissolution rate, chemical stability, absorption of drugs, and
conformational stabilization of proteins and lipids through encapsulation of their hydrophobic moieties.[22–27] This so-called
“molecular chaperone” effect of CDs has been used to stabilize the conformation of lipid A (a lipid component of an
endotoxin) adsorbed on PEM films.[24]
We present here the first PEM architecture that can drive
embryoid bodies (EBs) to cartilage and bone differentiation.
We selected a multilayer constructed from poly(L-glutamic
acid) (PLGA), poly(L-lysine) (PLL), and poly(L-lysine succinylated) (PLLs) films into which BMP2 and TGFb1 had been
embedded. We examined whether the BMP2 and TGFb1 activity could be retained and induce osteogenesis in ES cells
mediated by these growth factors embedded in a PEM film.
We found that both BMP2 and TGFb1 needed to be present
simultaneously in the film to drive the EBs to cartilage and
bone formation. In addition, this constitutes the first example
of a multilayer whose biological activity is based on a synergy
effect of two active compounds.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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The growth-factor transfer system we tested was based on
BMP2, TGFb1, and sequentially deposited monocarboxylic-bCD (cCD ). We investigated different architectures
(PLL-PLGA)10-PLL-(PLGA-PLL)5
(1)
(PLL-PLGA)10-PLL-(PLGA-PLLs)5
(2)
(PLL-PLGA)5-PLL-BMP2-PLL-(PLGA-PLLs)5
(3)
The presence of chondrocytes was recorded by Alcian-bluespecific staining of acidic proteoglycans after 10 days of EBs
growing on the surface of PEM films with simultaneous embedded BMP2 and TGFb1 (Architecture 5 or 6) (Fig. 1). Interestingly, no presence of chondrocytes was recorded by
using only embedded BMP2 or TGFb1 (Architecture 3 or 4),
or without BMP2 or TGFb1 (Architecture 2). Our results indi-
(PLL-PLGA)5-PLL-cCD-TGFb1-cCD-PLL-(PLGA-PLLs)5
(4)
(PLL-PLGA)5-PLL-cCD-TGFb1-cCD-PLL-(PLGA-PLL)5BMP2-PLL-(PLGA-PLLs)5
(5)
(PLL-PLGA)5-PLL-BMP2-PLL-(PLGA-PLL)5 cCD-TGFb1cCD-PLL-(PLGA-PLLs)5
(6)
These architectures were first analyzed by using nonsuccinylated PLL (Architecture 1). Our results clearly indicated nonadhesion of the EBs on the surface of this PEM film. Interestingly, by using succinylated PLLs (Architecture 2), we
increased the adhesion and the differentiation of the EBs
growing on the surface. It is expected that this was because of
an increase of the Young’s modulus of the film, which would
increase cellular adhesion.[28]
According to previous results obtained on similar PEMs
with embedded proteins[23] and DNA[29] it was expected that
the growth factors would remain confined in a layer structure
close to their deposition location. PEMs 5 and 6 were expected to have a somewhat stratified structure. The build up
of (PLLs-PLGA)5-PLLs film was monitored by using a
quartz-crystal microbalance (QCM). The increase in –Df/m
(the normalized frequency shift) with the number of deposited layers suggested regular film construction along the successive injections of PLLs and PLGA (Supporting information, Fig. S1).
During embryo development endochondral ossification
occurs in two steps: chondrocytes arise after mesenchymal
condensation and become hypertrophic, characterized by the
expression of collagen type II, and calcification. To test
whether this was true for the EBs growing on the surface of
(PLL-PLGA)10-PLL-(PLGA-PLLs)5 films, we analyzed the
differentiation of these EBs with and without embedded
BMP2 and TGFb1, cultured in the presence of insulin and
ascorbic acid, at 10 and 21 days of culture. Type II collagen
was found specifically in articular cartilage and was synthesized as a procollagen in two forms (IIA and IIB). Type IIA
collagen was expressed in juvenile or prechondrogenic cells,
whereas type IIB collagen was expressed in adult or differentiated chondrocytes. BMP2 and TGF-b1 induced chondrocytes
underwent hypertrophy and began to alter their expression
profile towards osteoblasts.
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Figure 1. Proteoglycans secreted by ES-cell derived chondrocytes were
stained with Alcian blue. The presence of chondrocytes was monitored
after the EB was 10 days old, and growing on the surface of a PEM film
with embedded BMP2 and TGFb1, by using phase-contrast microscopy
(20×).
cate clearly that the simultaneous presence of both BMP2 and
TGFb1 is needed for the proteoglycans induction. We evidence here that both factors acted by synergy, however our results do not depend on the order of the growth-factor embedding.
Our histological results clearly indicate that the PEM films
functionalized simultaneously with BMP2 and TGF-b1 provide an environment that promotes chondrogenic and osteogenic differentiation of ES-cell-derived EBs. It is also clearly
shown that both pathways of differentiation were stimulated
and enhanced by the two growth factors BMP2 and TGFb1. In
the presence of both factors, large cartilage nodules (Fig. 1)
were detected, whereas only some areas of cartilaginous structures could be stained without them.
Von Kossa staining for osteoprogenitors, on the 21st day of
EB growth in differentiation conditions, indicated clearly the
presence of mineralized structures in black (Fig. 2). EB-derived mineralized osteocalcin-expressing osteoblasts were detected as large areas of stained mineralized structures on the
surface of the (PLL-PLGA)5-PLL-cCD-TGFb1-cCD-PLL(PLGA-PLLs)5-BMP2-PLL-(PLGA-PLLs)5 films (Fig. 2B
and C), whereas only sporadic black-stained areas were visible on (PLLs-PLGA)10-PLL-(PLGA-PLLs)5 films (Fig. 2A).
To corroborate the role of BMP2 and TGFb1 in chondrogenesis and osteogenesis, we conducted tests by using a reverse-transcriptase polymerase chain reaction (RTPCR) for
the expression of chosen marker genes. Expression of
type IIB collagen and aggrecan, indicative of a fully mature
state of cartilage, as well as the osteogenic markers osteocalcin and osteopontin were measured at 10 and 21 days of EB
culture in our differentiating conditions (Fig. 3). Figure 3
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2007, 19, 693–697
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one of the two growth factors does not
induce any differentiation. After chondrogenesis for up to 10 days of EB
culture, a switch towards bone formation occurred. It was characterized,
after 21 days of culture, by the specific
expression of the osteocalcin and
osteopontin genes, as well as by the
presence of mineralized cells highFigure 2. Von Kossa staining of EBs differentiated on the surface of a (PLLs-PLGA)10-PLL-(PLGAlighted by the specific von Kossa stainPLLs)5 film (A) or a (PLL-PLGA)5-PLL-cCD-TGFb1-cCD-PLL-(PLGA-PLL)5-BMP2-PLL-(PLGA-PLLs)5
ing (Fig. 2B and C).
film (B,C).
We also analyzed the expression of
osteopontin by using immunocytochemistry of EBs, differentiated to the osteoblast lineage, growing
Marker
on the surface of PEM films functionalized or not with BMP2
and TGF-b1 (Fig. S2, Suporting Information). Our results indicate clearly the specific expression of osteopontin induced
when BMP2 and TGF-b1 were simultaneously embedded into
1000
PEM film.
500
During the three major phases of osteogenesis, i) proliferation, ii) extracellular-matrix (ECM) deposition and maturation, iii) mineralization and the expression pattern of typical
100
markers is organized temporally and sequentially. Our results
demonstrate clearly that we are able to induce osteogenesis in
ES cells mediated by growth factors embedded in a PEM film.
1 2 3 4 5 6 7 8 9 101112131415
We are able to induce undifferentiated ES cells in vitro to
10 days 21 days
WT
the osteoblastic phenotype at high efficiency, expressing all
of EB differentiation D3 ES cells
major genes specific for mineralization processes during
Figure 3. A RTPCR analysis of the expression of osteochondral extracelluosteogenesis.
lar matrix components (aggrecan, type IIB collagen, osteocalcin, and osA final study using electron microscopy indicated clearly
teopontin) in D3 EBs cultured for 10 or 21 days on multilayer films functhe cartilage induction (observed by using scanning electron
tionalized with BMP2 and TGFb1 in the presence of ascorbic acid and
microscopy (SEM); Fig. 4), and the bone induction (observed
insulin, compared to wild-type D3 ES cells. At the indicated differentiaby transmission electron microscopy (TEM) and visualized by
tion times, RNA was extracted and screened by using a RTPCR for hypoxanthine-guanine phosphoribosyl transferase (HPRT): H = 507 base pairs
bone vesicle induction and osteoblast differentiation; Fig. 5).
(bp) (internal control lanes 1, 6, 11); Aggrecan: AG = 270 bp (lanes 2, 7,
As far as the mechanism of action is concerned, one can
12); Collagen form II: CO = 432 bp (lanes 3, 8, 13); Osteocalcin:
imagine
two possibilities: i) the growth factors could be
OC = 370 bp (lanes 4, 9, 14); Osteopontin: OP = 460 bp (lanes 5, 10, 15).
Expression of both genes measured at earlier states is not excluded (and
not tested here). It might be much higher because our microscopic results are representative for mature protein markers.
summarizes the results from three independent differentiation
experiments. At 10 days of culture, specific bands showed
expression for aggrecan and collagen type IIB. The faint intensity of both bands reflects the available quantity of mRNA
at this particular time chosen for differentiation.
A similar result was obtained at 21 days of culture where
mineralized cells might represent mature osteoblasts. A very
faint, but specific band for osteocalcin was always seen in all
experiments, whereas the level of osteopontin expression in
all samples tested was always very high compared to the expression of hypoxanthine–guanine phosphoribosyl transferase
(HPRT) used as internal control. HPRT expression was at the
same level in differentiated and wild-type ES cells.
Our results indicate clearly that it is possible to differentiate
EBs on the PEM film functionalized simultaneously with
BMP2 and TGF-b1. One can note that the presence of only
Adv. Mater. 2007, 19, 693–697
Figure 4. SEM observation of cartilage induction (collagen induction) by
EBs growing on the surface of (PLL-PLGA)5-PLL-cCD-TGFb1-cCD-PLL(PLGA-PLL)5-BMP2-PLL-(PLGA-PLLs)5 films (Scale bars in A: 20 lm,
B: 10 lm, C: 5 lm, and D: 2 lm).
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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scaffolds, coated by embedded growth factors, sufficient to
repair large-scale bone defects and to determine if viable
grafts can be obtained from both ES and ES-derived material.
Experimental
Figure 5. TEM observation of bone induction by EBs growing on the surface of (PLL-PLGA)5-PLL-cCD-TGFb1-cCD-PLL-(PLGA-PLL)5-BMP2-PLL(PLGA-PLLs)5 films. A) Ossification visualized in black on the collagen
fibers; B) bone vesicle induction; C) osteoblast visualization.
released from the film and interact with cells through the solution or ii) the growth factors could come into contact with the
cells through the multilayer. Control experiments were performed in which cells were incubated in contact with supernatant from similar architectures. No differentiation was
observed. Moreover, when cells were deposited on unfunctionalized PEM film, in contact with solutions containing both
growth factors, only a very weak differentiation was observed
compared to that for embedded growth factors. These observations support the hypothesis that the cells came into contact
with the growth factors through the PEM films. This could be
because of film degradation by the cells or by diffusion of the
growth factor through the PEMs up to the surface. Based on
current knowledge, one cannot distinguish between the two
possibilities. However, previous investigations on biofunctionalization of similar PEMs always showed that cells came into
contact with the active compounds (proteins, DNA) through
cellular-film degradation. A complete investigation of the
molecular mechanism of cellular interaction is however out of
the scope of this Communication.
These findings suggest that it is possible to differentiate
EBs on the PEM film functionalized with appropriate growth
factors. We present here for the first time the fact that simultaneously embedded BMP2 and TGFb1 in a PEM film can
drive ES cells to cartilage or bone differentiation. Moreover,
this constitutes the first example of a bifunctionalized multilayer where a synergy effect between the embedded active
compounds is necessary to induce the biological response.
These results also indicate clearly that a biomaterial coated
with multilayer films that interact with cells by inducing specific differentiation depending on the embedded active molecules is possible.
In order to enable the efficient repair of bone under the
suboptimal conditions found in aged or diseased patients, we
would ideally engraft populations of cells already committed
to osteogenic (bone) lineage. Our broad long-term objective
is to provide combinations of osteoprogenitor cells and active
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Chemicals: PLL hydrobromide (molecular weight, MW = 30.3 kDa),
PLLs (MW = 50 kDa, 35 % modification of the amine residues), PGA
(MW = 54 kDa), were purchased from Sigma (St. Quentin Fallavier,
France). BMP2 and TGFb1 were from R&D system. Bisbenzimide
H 33258 (Hoechst) used for microscopy was purchased from Invitrogen. cCD [22] was from R. Darcy (UCD, Dublin).
Polyelectrolyte Multilayer Film Preparation: For all in vitro experiments, PEM films were prepared on glass coverslips (CML, France)
pretreated with 10–2 M sodium dodecyl sulfate (SDS) and 0.12 N HCl
for 15 min at 100 °C, and then extensively rinsed with deionized
water. Glass coverslips were deposited in 24-well plates (Nunc, Denmark). Films fabricated from n(PLL-PLGA) bilayers were built by alternate immersion of the pretreated coverslips for 10 min in polyelectrolyte solutions (300 lL) at a concentration of 1 mg mL–1 for PLLs
and PLGA in the presence of 0.15 M NaCl at pH 7.4 for each solution
(300 lL). These were added to polyanion-ending architectures for 1 h
and the buildup was continued by the further addition of layers. After
each deposition the coverslips were rinsed three times for 10 min with
0.15 M NaCl. All the films were sterilized for 10 min by UV light
(254 nm). Before use, the architectures were put in contact with 1 mL
of cell-culture medium without serum for 24 h.
Quartz-Crystal Microbalance: The film buildups were monitored
in situ by using a QCM using an axial flow chamber QAFC 302
(QCM-D, D300, Q-Sense, Götenborg, Sweden). This QCM technique
consists of measuring the resonance frequency shift (Df) of a quartz
crystal induced by a polyelectrolyte or protein adsorption on the crystal, compared to the crystal in contact with a buffer. Changes in the
resonance frequency were measured at the third overtone (m = 3) corresponding to the 15 MHz resonance frequency. A shift in Df/m can be
associated, to a first approximation, to a variation of the mass
adsorbed to the crystal through the Sauerbrey relation [30]: mass,
m = –C Df/m, where C is a constant that is characteristic of the crystal
used (C = 17.7 ng cm–2 Hz–1). The measurement methodology has been
addressed in detail elsewhere and is applied in the present work [31].
Cell Culture and Differentiation of Embryonic Stem Cells: The
mouse ES cell line D3 (gift from R. Kemler) was kept undifferentiated as described [11]. To induce differentiation, the ES cells, free of
feeder cells by extensive plating, were cultured in hanging drops
(1000 cells/30 lL) for 48 h in ES medium without leukemia inhibitory
factor (LIF). The ES medium was supplemented with insulin
(1 lg mL–1) and ascorbic acid (50 lg mL–1), and the fetal bovine serum was increased to 20 %. The formed EBs were maintained in suspension from day 3 to 5 in ultralow-adherent culture dishes (Stem cell
technologies Inc) and then plated on coated coverslips into 4- or
24-well tissue culture plates. The effects of BMP2 (10 ng mL–1),
TGFb1 (2 ng mL–1), in various combinations on the differentiation for
chondrocytes and osteoblasts were examined. The medium was
changed every second day.
Alcian-Blue Staining: Proteoglycans secreted by ES-cell derived
chondrocytes were stained with Alcian blue. Cultures were fixed in
2.5 % glutaraldehyde, 25 mM sodium acetate, and 0.4 M MgCl2 containing 0.05 % Alcian blue for 48 h. Wash steps in 3 % acetic acid,
3 % acetic acid/25 % ethanol, and 3 % acetic acid/50 % ethanol reduced unspecific binding of the dye. The presence of chondrocytes
was monitored by phase-contrast microscopy (20×).
Von Kossa Staining for Osteoprogenitors: The differentiated cells
were washed twice with phosphate-buffered saline (PBS), and then
fixed for 2 h at room temperature (RT) with 10 % neutral-buffered
formalin. After washing with distilled H2O (dH2O), the cells were
stained for 30 min with 2.5 % silver nitrate (freshly prepared). After
two washes with dH2O, cells were counterstained 10–15 s with 0.1 %
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2007, 19, 693–697
Received: June 9, 2006
Revised: September 27, 2006
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toluidine blue, washed again three times with dH2O and air-dried.
The presence of mineralized structures (in black) was recorded by
using a cool snap camera coupled to a Leica DRBH microscope.
Immunofluorescence for Osteopontin Expression: EBs growing on
the surface of multilayered film (PLLs-PGA)5-PLLs-cCD-TGFb1cCD-(PLLs-PGA)5-PLLs-BMP2-(PLLs-PGA)5-PLLs) were differentiated to the osteoblast lineage. As a control, EBs were differentiated
on gelatinized coverslips in presence of BMP2, TGFb1, insulin, and
ascorbic acid, and fixed on day 21 with 2 % paraforaldehyde. Immunocytochemistry was carried out as earlier described [31]. Cells were
first treated with a monoclonal mouse antiosteopontin antibody
(OPN (Akm2A1): sc-21742; Santa Cruz Biotechnology). The second
antibody was a Cy3 goat antimouse immunoglobulin (IgG; heavy and
light chains). Counterstaining of the cells was carried out by means of
a 20 s Hoechst treatment (5 ng mL–1). Immunostaining for osteopontin expressing cells (red) was monitored by using a cool snap camera
coupled to a Leica DRB microscope using a specific CY3 filter.
Reverse-Transcriptase Polymerase Chain Reaction: Total RNA of
wild-type and differentiated ES cells (cultured in the presence or absence of BMP2 and TGFb1) was extracted using the RNeasy Micro
Kit (Qiagen) according to the manufacturer’s instructions. 2 lg of total RNA were reverse-transcribed in the presence of 200 ng of hexamer primer, 0.1 M dithiothreitol (DTT), 40 units of RNAsine (an RNase
inhibitor), deoxyribonucleate triphosphates (dNTPs) (2.5 mM each),
Montana myotis leukoencephalitis virus (MMLV) transcriptase
(500 units), and reaction buffer for 1 h at 37 °C. 2 lL of the reversetranscriptase products were used for the different polymerase chain
reactions. The polymerase chain reactions were cycled (30 cycles) by
denaturing for 30 s at 95 °C, annealing for 30 s at 60 °C, and elongation for 1 min at 72 °C, with a final elongation for 10 min at 72 °C.
The primer sequences were as follows: HPRT (507 base pairs (bp))
5′-GCCTGTATCCAACACTTCG-3′ and 5′-AGCGTCGTGATTAGCGATG-3′ [32], Collagen II: (variant A: 432 bp; variant B: 225 bp)
5′-AGGGGTACCAGGTTCTCCATC-3′ and 5′-CTGCTCATCGCCGCGGTCCTA-3′ [33], Osteocalcin (370 bp) 5′-CAAGTCCCACACAGCAGCTT-3′ and 5′-AAAGCCGAGCTGCCAGAGTT-3′
[34], Osteopontin (460 bp) 5′-CTGGCTTTGGAACTTGCTTGAC-3′
and 5′-CGACGATGATGACGATGATGAT-3′ [35], and Aggrecan
(270 bp) 5′-TCCTCTCCGGTGGCAAAGAAGTTG-3′ and 5′-CCAAGTTCCAGGGTCACTGTTACCG-3′ [36]. PCR products were
analyzed on a 2 % agarose gel.
Histological and Electron Microscopy Analysis: The samples were
fixed in Karnovsky fixative, post-fixed with 1 % osmium tetroxide in
0.1 M cacodylate buffer for 1 h at 4 °C, dehydrated through graded
alcohol and embedded in Epon 812. Semithin sections were cut at
2 lm and stained with toluidine blue, and histologically analyzed by
using light microscopy. Ultrathin sections were cut at 70 nm and contrasted with uranyl acetate and lead citrate, and examined with a
Philips 208 electron microscope. For SEM, samples were fixed, dehydrated as above, dried with critical-point drying apparatus, and then
mounted on aluminum stubs coated with palladium–gold using a cold
sputter-coater and observed with a Philips XL-20 microscope.
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